Redirection of tropism of aav capsids

ABSTRACT

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of: U.S. Provisional Patent Application No. 62/740,310, filed Oct. 2, 2018, entitled AAV CAPSID LIBRARIES AND TISSUE TARGETING PEPTIDE INSERTS; U.S. Provisional Patent Application No. 62/839,883, filed Apr. 29, 2019 entitled REDIRECTION OF TROPISM OF AAV CAPSIDS; the contents of which are each incorporated herein by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20571060PCTSL.txt, created on Oct. 2, 2019, which is 428,491 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.

BACKGROUND

Gene delivery to the adult central nervous system (CNS) remains a major challenge in gene therapy, and engineered AAV capsids with improved brain tropism represent an attractive solution.

Adeno-associated virus (AAV)-derived vectors are promising tools for clinical gene transfer because of their non-pathogenic nature, their low immunogenic profile, low rate of integration into the host genome and long-term transgene expression in non-dividing cells. However, the transduction efficiency of AAV natural variants in certain organs is too low for clinical applications, and capsid neutralization by pre-existing neutralizing antibodies may prevent treatment of a large proportion of patients. For these reasons, major efforts have been devoted to obtaining novel capsid variants with enhanced properties. Of many approaches tested so far, the most significant advances have resulted from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by capsid sequence randomization using either error-prone PCR, shuffling of various parent serotypes or insertion of fully randomized short peptides at defined positions.

In order to perform directed evolution of AAV capsids, the sequence encoding the viral capsid is itself flanked by inverted terminal repeats (ITR) so it can be packaged into its own capsid shell. Following infection of cultured cells or animals by the mixed population of capsids, the DNA encoding capsids variants that have successfully homed into the tissue of interest is recovered by PCR for further rounds of selection. In this approach, all viral DNA species present in a given tissue are recovered, with no discrimination for specific cell types or for vectors able to perform complete transduction (cell surface binding, endocytosis, trafficking, nuclear import, uncoating, second-strand synthesis, transcription). For example, in the case of highly complex tissues containing multiple cell types, such as the central nervous system (CNS), it would be highly preferable to apply a more stringent selective pressure aimed at recovering capsid variants capable of transducing neuron and/or astrocyte rather than microglia or blood vessel endothelial cells.

Attempts at improving the CNS tropism of AAV capsids upon systemic administration have been met with limited success.

Two previous approaches have been used to address this issue. The first strategy used co-infection of cultured cells (Grimm et al., 2008) or in situ animal tissue (Lisowski et al., 2014) with adenovirus, in order to trigger exponential replication of infectious AAV DNA. Another successful approach involved the use of cell-specific CRE transgenic mice (Deverman et al., 2016) allowing viral DNA recombination specifically in astrocytes, followed by recovery of CRE-recombined capsid variants. Both approaches proved successful, allowing the isolation of several capsid variants with enhanced transduction of target cell populations.

This finding suggested that cell type-specific library selection could improve the outcome of directed evolution. However, the transgenic CRE system used by Deverman et al. is not tractable in other animal species and AAV variants selected by directed evolution in mouse tissue do not show similar properties in large animals. Therefore, it would be necessary to perform the entire directed evolution process directly in non-human primates to increase the probability of translatability in human subjects. None of the previously described transduction-specific approaches are amenable to large animal studies because: 1) many tissues of interest (e.g. CNS) are not readily accessible to adenovirus co-infection, 2) the specific Ad tropism itself would bias the library distribution, and 3) large animals are typically not amenable to transgenesis and cannot be genetically engineered to express CRE recombinase in defined cell types.

To address this problem, we have developed a broadly-applicable functional AAV capsid library screening platform for cell type-specific biopanning in non-transgenic animals. In the TRACER (Tropism Redirection of AAV by Cell type-specific Expression of RNA) platform system, the capsid gene is placed under the control of a cell type-specific promoter to drive capsid mRNA expression in the absence of helper virus co-infection. This RNA-driven screen increases the selective pressure in favor of capsid variants which transduce a specific cell type.

The TRACER platform allows generation of AAV capsid libraries whereby specific recovery and subcloning of capsid mRNA expressed in transduced cells is achieved with no need for transgenic animals or helper virus co-infection. Since mRNA transcription is a hallmark of full transduction, these methods will allow identification of fully infectious AAV capsid mutants. In addition to its higher stringency, this method allows identification of capsids with high tropism for particular cell types using libraries designed to express CAP mRNA under the control of any cell-specific promoter such as, but not limited to, synapsin-1 promoter (neurons), GFAP promoter (astrocytes), TBG promoter (liver), CAMK promoter (skeletal muscle), MYH6 promoter (cardiomyocytes).

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for the engineering and/or redirecting the tropism of AAV capsids. Also provided herein are peptides which may be inserted into AAV capsid sequences to increase the tropism of the capsid for a particular tissue. In one aspect, the peptides may be used to target the capsids to brain or regions of the brain or the spinal cord.

The present disclosure presents methods for generating one or more variant AAV capsid polypeptides. In certain embodiments, the variant AAV capsid polypeptides exhibit at least one of improved transduction or increased cell or tissue specificity, relative to a parental AAV capsid polypeptide. In certain embodiments, the method includes: (a) generating a library of variant AAV capsid polypeptides, wherein said library includes (i) a plurality of capsid polypeptides having a region of randomized sequence of 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids, or (ii) a plurality of capsid polypeptides from more than one parental AAV capsid polypeptide; (b) generating an AAV vector library by cloning the capsid polypeptides of libraries (a)(i) or (a)(ii) into AAV vectors, wherein the AAV vectors include a first promoter and a second promoter, wherein said second promoter drives capsid mRNA expression in the absence of helper virus co-infection.

In certain embodiments, the first promoter is AAV2 P40. In certain embodiments, the second promoter is a ubiquitous promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.

In certain embodiments, the first promoter is AAV2 P40. In certain embodiments, the second promoter is a cell-type-specific promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a cell-type-specific promoter.

In certain embodiments, the promoter is selected from any promoter listed in Table 3. In certain embodiments, the ubiquitous or cell-specific promoter allows the expression of RNA encoding the capsid polypeptides.

In certain embodiments, the method includes recovery of the RNA encoding the capsid polypeptides. In certain embodiments, the method includes determining the sequence of the capsid polypeptides. In certain embodiments, the capsid polypeptides recovered exhibit increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

In certain embodiments, the target cell is a neuronal cell, a neural stem cell, an astrocyte, an oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell.

In certain embodiments, the AAV vectors comprise a first promoter and a second promoter, wherein the second promoter is located the downstream of the capsid gene and drives its anti-sense RNA expression in the absence of helper virus co-infection.

In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter. In certain embodiments, the first promoter is AAV2 P40 and the second promoter is a cell-specific promoter. In certain embodiments, the ubiquitous or cell-specific promoter allows the expression of gene encoding the capsid polypeptide of variant AAV in an anti-sense direction, resulting in the anti-sense RNA. In certain embodiments, the method included the recovery of the anti-sense RNA that can be converted to RNA encoding the variant AAV capsid polypeptide that is used to determine the sequence of the variant AAV capsid polypeptides.

In certain embodiments, the variant AAV capsid polypeptide exhibits increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages will be apparent from the following description of particular embodiments of the disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure.

FIG. 1A and FIG. 1B are maps of wild-type AAV capsid gene transcription and CMV-CAP vectors. FIG. 1A shows transcription of VP1, VP2 and VP3 AAV transcripts from wildtype AAV genome. Transcription start sites of each viral promoter are indicated. SD, splice donor, SA, splice acceptor. Sequence of start codons for each reading frame is indicated. Translation of AAP and VP3 is performed by leaky scanning of the major mRNA. FIG. 1B shows the structure of the CMV-p40 dual promoter vectors used to determine the minimal regulatory sequences necessary for efficient virus production. The pREP2ΔCAP vector shown at the bottom is obtained by deletion of most CAP reading frame and is used to provide the REP protein in trans.

FIG. 2A and FIG. 2B are histogram representations of the data and show the effect of CMV promoter position on virus yield and CAP mRNA splicing. FIG. 2A shows average yield of AAV9 produced in HEK-293T cells using the constructs described in FIG. 1, co-transfected with an Ad Helper vector. Wild-type AAV9 plasmid (pAV9) is used as a positive control. Y-axis values indicate AAV DNA copies per ul from each 15-cm plate (˜1000 ul total, left panel) or the percentage of wtAAV9 (right panel). FIG. 2B shows evidence for expression of CAP transcripts in transfected cells. mRNA from transfected 293T cells was subjected to RT-PCR using primers specific for the major spliced CAP transcript. Note the lack of p40-driven transcription in the absence of Ad Helper vector (lane 2).

FIG. 3A, FIG. 3B and FIG. 3C show the effect of REP helper plasmid optimization on virus yield. FIG. 3A shows the design of improved pREP helper vectors. The MscI fragment deletion removes the C-terminal part of VP proteins, which is necessary for capsid formation. Asterisks represent early stop codons introduced to disrupt the coding potential of VP1, VP2 and VP3 reading frames. FIG. 3B shows the yield of Synapsin-p40-CAP9 AAV produced with various REP plasmid architectures. Values on the Y-axis represent the percentage of VG relative to wild-type AAV9. FIG. 3C shows the quantification of recombination and/or illegitimate packaging of full-length REP from the pREP plasmids. Virus stocks produced were subjected to qPCR using Taqman probes located in the N-terminal part of REP absent from the ITR-containing vectors.

FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D describe the in vivo analysis of the second-generation vectors. FIG. 4A shows the design of Pro9 vectors. Architecture of all three vectors is based on the BstEII construct. AAV9 capsid RNA is placed under control of P40 and CMV, hSyn1 or GFAP promoters, respectively. FIG. 4B shows the silver stain of SDS-PAGE gel obtained by running 1e10 VG of each vector, after double iodixanol purification. FIG. 4C shows the biodistribution of viral DNA in mouse brain (cortex), liver and heart following tail-vein injection of 1e12 VG per mouse. AAV9 VP3 DNA is quantified by Taqman PCR and normalized to mouse transferrin receptor gene. FIG. 4D shows the capsid RNA recovery from mouse tissues. Total RNA was reverse transcribed and Taqman PCR was performed with capsid-specific Taqman primers and probe. Values represent VP3 cDNA copies normalized to TBP housekeeping gene.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E describes in vitro analysis of intronic second generation vectors. FIG. 5A shows the design of intronic Pro9 vectors harboring a hybrid CMV/Globin intron. AAV9 capsid RNA is placed under control of P40 and CBA, hSyn1 or GFAP promoters in a tandem configuration (top) or in an inverted configuration (bottom). In the inverted promoter vectors, an extra SV40 polyadenylation site (orange) is added at the 3′ extremity to allow polyadenylation of antisense CAPS transcripts. FIG. 5B shows the AAV9 CAP cDNA amplification. All vectors depicted were produced using triple transfection with pHelper and pREP-3stops and resulting viruses were used to infect HEK-293T cells at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-infection and subjected to RT-PCR with primers amplifying full capsid (top) or a C-terminal fragment (bottom). FIG. 5C shows the AAV9 VP3 cDNA from cells infected with intronless or intronic viruses with tandem promoters in forward orientation was quantified by Taqman PCR and normalized to GAPDH housekeeping gene. Values indicate the ratio of VP3 to GAPDH cDNA. FIG. 5D shows the mapping of capsid RNA recovery from cells infected with tandem or inverted constructs. Total RNA was reverse transcribed and PCR was performed with primers flanking the entire capsid gene. White arrowheads represent VP3 size variants resulting from aberrant splicing of antisense CAP mRNA. FIG. 5E shows the analysis of Globin intron splicing. CAG9 plasmid (left) or cDNA from HEK-293T cells transduced by CAG9 virus was submitted to PCR with forward primers located before (Glo ex1) or within (GloSpliceF4 (SEQ ID NO: 26) and GloSpliceF6 (SEQ ID NO: 13)) the Globin exon-exon junction. Primers spanning junction between exon 1 (no underline) and exon 2 (underline) are described at the bottom.

FIG. 6 provides in vitro evidence that the presence of the P40 promoter downstream of Synapsin or Gfabc1D promoters does not relieve the repression of either promoter in HEK-293T cells.

FIG. 7 illustrates the basic tenets of the TRACER platform.

FIG. 8 illustrates features of the TRACER platform including the use of a tissue specific promoter and RNA recovery.

FIG. 9 provides one embodiment of the TRACER production architecture.

FIG. 10 provides a comparison between traditional vDNA recovery and 2^(nd) generation vRNA recovery.

FIG. 11 provides an overview of the use of cell-specific RNA expression for targeted evolution.

FIG. 12A and FIG. 12B provide diagrams representing capsid gene transcription of natural AAV (FIG. 12A) and TRACER libraries (FIG. 12B).

FIG. 13 is a diagram of the AAV6, AAV5 and AAV-DJ capsid peptide display libraries used for in vivo evolution (SEQ ID NOS 27-32, respectively, in order of appearance).

FIG. 14 is a diagram of the AAV9 capsid peptide display libraries used for in vivo evolution (SEQ ID NOS 33-42, respectively, in order of appearance).

FIG. 15A and FIG. 15B present the method used for library construction. FIG. 15A shows the sequence of the insertion site used to introduce random libraries (SEQ ID NOS 43-46, respectively, in order of appearance). FIG. 15B provides a description of the assembly procedure.

FIG. 16 provides an exemplary diagram of cloning-free rolling circle procedure used for library amplification (SEQ ID NO 47; NNK₇).

FIG. 17 provides the sequence of the codon-mutant AAV9 library shuttle designed to minimize wild-type contamination (SEQ ID NOS 33-34 and 48-52, respectively, in order of appearance).

FIG. 18 provides a description of AAV9 peptide libraries biopanning.

FIG. 19 illustrates the recovery process from an initial pool with recovery at 50%.

FIG. 20 provides an example of the cDNA recovery and amplification from GFAP-driven libraries (B group and F group).

FIG. 21A, FIG. 21B and FIG. 21C show the progression of AAV9 peptide library diversity throughout the biopanning process. FIG. 21A describes RNA library evolution. FIG. 21B and FIG. 21C show the amino acid distribution of NNK machine mix preparations for P0 and P1 virus.

FIG. 22 provides neuron (SYN)-AAV9 Peptide Libraries Composition at P2.

FIG. 23 provides astrocyte (GFAP)-AAV9 Peptide Libraries Composition at P2.

FIG. 24 provides an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

FIG. 25 provides an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

FIG. 26 provide an example subpopulation selection of variants.

FIG. 27 provides an exemplary design of a library generation and cloning procedure.

FIG. 28 provides the NNK/NNM codon distribution (covariance of codon mutants) of AAV produced with a synthetic library of 666 sequence variants (GFAP promoter).

FIG. 29 provides the NNK/NNM codon distribution (covariance of codon mutants) of AAV produced with a synthetic library of 666 sequence variants (SYN9 promoter).

FIG. 30 provides the data from the tissue recovery, one-month post injection, from brain and a liver punch.

FIG. 31A, FIG. 31B, FIG. 31C and FIG. 31D provide results of control capsids from the Syn-driven synthetic library NGS analysis. FIG. 31A shows the enrichment analysis of internal AAV9, PHP.B and PHP.eB controls (SEQ ID NOS 53-58 and 53-58, respectively, in order of appearance). FIG. 31B, FIG. 31C and FIG. 31D show the NNK/NNM codon distribution in mRNA from mouse brain tissue.

FIG. 32A and FIG. 32B provide the results of the neuron synthetic library NGS analysis (SEQ ID NOS 59-60, 59-61, 61-63, 62, 64, 64, 63, 65-67, 67, 65, 68, 66, 69, 70-71, and 70-74, respectively, in order of appearance).

FIG. 33 provides the results of the astrocyte synthetic library NGS analysis (SEQ ID NOS 53-58, 53-58, and 53-58, respectively, in order of appearance).

FIG. 34A and FIG. 34B provide astrocyte synthetic library codon mutants covariance.

FIG. 35 provides the results of the astrocyte synthetic library NGS analysis (SEQ ID NOS 75, 75-78, 76-77, 79-83, 65, 78, 84, 80, 85, 70, 86, 82, 81, 79, 87, 65, 85, 84, 70, 86, 88-90, 87, 91, 83, 88, 63, 89-90, 92-93, 91, 94-97, 93, 95, 98, 98, 97, 63, 92, 94, 99-101, 75, 75-78, 76-77, 79-83, 65, 78, 84, 80, 85, 70, 86, 82, 81, 79, 87, 65, 85, 84, 70, 86, 88-90, 87, 91, 83, 88, 63, 89-90, 92-93, 91, 94-97, 93, 95, 98, 98, 97, 63, 92, 94, 99-102, 99, 103, 103-104, 96, 105-106, 101, 100, 102, 107, 104-105, 108-113, 106, 60, 66, 114-117, 109, 113, 72, 108, 110, 67, 118-119, 116, 120, 120, 107, 112, 121-123, 66, 124-125, 115, 118, 126, 121, 127-128, 60, 129, 119, 130-132, 72, 133, 123, 125, 69, 134-139, 62, 124, 67, 111, 114, 126, 140-141, 122, 142, 128-129, 143, 138, 144, 134, 62, 136, 145, 141, 146-153, 127, 154, 69, 144, 155, 71, 156, 133, 132, 137, 147, 157-158, 135, 159, 140, 117, 160, 139, 161-162, 130, 163, 143, 164, 152, 151, 165-167, 155, 168, 71, 169, and 146, respectively, in order of appearance).

FIG. 36 provides the GFAP synthetic library NGS analysis.

FIG. 37A and FIG. 37B provides the top 38 variants from the synthetic library screen. FIG. 37A shows the phylogenetic analysis of 9-mer peptide sequences, and also shows the sequence of the peptide variants (SEQ ID NOS 67, 59, 64, 61, 77, 84, 96, 60, 80, 82, 66, 62, 83, 85, 106, 131, 94, 90, 76, 68-69, 79, 75, 81, 88, 139, 78, 155, 102, 63, 140, 87, 70, 105, 120, 89, 65, and 109, respectively, in order of appearance). Highlighted sequences represent the peptides that were selected for individual transduction assay. FIG. 37B shows the graphic representation of the neuron and astrocyte tropism of each peptide, both axis indicate the inverted rank in Synapsin and GFAP screen.

FIG. 38 provides the top consensus sequences as compared to PHP.N and PHP.B (SEQ ID NOS 168 and 71, respectively, in order of appearance).

FIG. 39 is a diagram of the Gibson assembly library cloning procedure.

FIG. 40 provides an example of TRIM/NNK peptide prevalence (SEQ ID NOS 170-171, respectively, in order of appearance).

FIG. 41 provides peptide diversity statistics from a study using the Illumina adapter having 42 million bacterial transformants, 81 million sequence reads and 12 million sequence variants (SEQ ID NOS 172-173, 48-49, and 174-175, respectively, in order of appearance).

FIG. 42 provides an exemplary diagram of cloning-free DNA amplification by rolling circle amplification.

FIG. 43 provides a diagram of protelomerase monomer processing (SEQ ID NOS 176-178, respectively, in order of appearance).

FIG. 44 provides a diagram comparing the traditional and cloning-free methods.

FIG. 45A and FIG. 45C provide the full ranking of Syn-driven (FIG. 45A) and GFAP-driven (FIG. 45B) 333 variants in the brain, spinal cord, liver and heart tissues. Capsid variants are ranked by their average brain RNA enrichment score (average of NNK and NNM codons). The rank of internal control capsids PHP.B, PHP.eB and AAV9 is indicated (FIG. 45A and FIG. 45B). A comparison of combined Syn-driven results and GFAP-driven results is provided (FIG. 45C). Only 4 animals were represented for the GFAP-driven libraries because 2/6 mice showed a very different ranking profile and were considered as outliers.

FIG. 46A and FIG. 46B provide the comparison of results of the neuron and astrocyte synthetic library NGS analysis. FIG. 46A shows the ranking of capsids using SYN or GFAP promoters; FIG. 46B shows the scatter plot showing the correlation of Syn-versus GFAP-driven libraries.

FIG. 47 illustrates one embodiment of a multi-species (e.g., rodent) study followed by next generation sequencing (NGS).

FIG. 48A, FIG. 48B and FIG. 48C provide results from a multi-strain/species comparison of 333 capsid variants. FIG. 48A shows the ranking of 333 capsids by brain RNA enrichment score in C57BL/6 mice, BALB/C mice and rats. Capsids are ranked according to Syn-driven brain enrichment score in C57BL/6 mice. FIG. 48B shows the scatter plots showing the correlation between C57BL/6 and BALB/C enrichment scores from Syn- and GFAP-driven pools. FIG. 48C shows the Venn diagram showing the intersection and consensus sequence of capsids with a brain enrichment score >10-fold higher than AAV9 (either Syn- or GFAP-driven) in C57BL/6 and BALB/C strains. In rats, no capsid showed an enrichment score >10-fold versus AAV9.

FIG. 49A, FIG. 49B, FIG. 49C and FIG. 49D provide transduction (RNA) and biodistribution (DNA) analysis of 10 capsid variants indicated in FIG. 49A (SEQ ID NOS 179-188, respectively, in order of appearance). Individual capsids were used to package self-complementary CBA-EGFP genomes (FIG. 49B) and injected intravenously to C57BL/6 mice. FIG. 49C shows the RNA expression in brain and spinal cord samples. FIG. 49D shows the DNA distribution in brain and spinal cord samples.

FIG. 50A, FIG. 50B and FIG. 50C provide the results of testing of individual capsids and their mRNA expression in brain, spinal cord and liver. EGFP mRNA expression results are shown for the brain (FIG. 50A), the spinal cord (FIG. 50B) and the liver (FIG. 50C).

FIG. 51 provides results for NGS screening using neuronal NeuN marker (FIG. 51) for both GFAP screening and SYN screening.

FIG. 52 provides the results of testing of individual capsids in whole brain.

FIG. 53 provides the results of testing of additional individual capsids in whole brain.

FIG. 54 provides the results of testing of individual capsids in cerebellum.

FIG. 55 provides the results of testing of individual capsids in cortex.

FIG. 56 provides the results of testing of individual capsids in hippocampus.

FIG. 57A and FIG. 57B provide transduction data of 10 capsid variants in mouse liver (FIG. 57B), analyzed by EGFP RNA expression and whole tissue fluorescence (FIG. 57A).

FIG. 58A and FIG. 58B provide results for comparison studies on the efficacy of the 333 capsid variants to transduce CNS for C57BL/6 mice BMVEC (FIG. 58A) and Human BMVEC (FIG. 58B).

FIG. 59A, FIG. 59B and FIG. 57C provide diagrams of external barcoding for NGS analysis and recovery of full-length capsid variants. A general barcode pair is shown (FIG. 59C). Full ITR-to-ITR constructs are shown with the barcode pair 5′ of the CAP sequence (FIG. 59A) and 3′ of the CAP sequence (FIG. 59B).

FIG. 60A, FIG. 60B and FIG. 60C provide detailed analysis of virus production and RNA splicing with several configurations of intronic barcoded platforms. A general ITR-to-ITR construct is shown in FIG. 60A (SEQ ID NOS 189-193, respectively, in order of appearance), with intronic barcode yields (FIG. 60B) and gel columns showing AAV intron splicing and Globin intron splicing results (FIG. 60C).

DETAILED DESCRIPTION OF THE DISCLOSURE

The details of one or more embodiments of the disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control.

According to the present disclosure, AAV particles with enhanced tropism for a target tissue (e.g., CNS) are provided, as well as associated processes for their targeting, preparation, formulation and use. Targeting peptides and nucleic acid sequences encoding the targeting peptides are provided. These targeting peptides may be inserted into an AAV capsid protein sequence to alter tropism to a particular cell-type, tissue, organ or organism, in vivo, ex vivo or in vitro.

As used herein, an “AAV particle” or “AAV vector” comprises a capsid protein and a viral genome, wherein the viral genome comprises at least one payload region and at least one inverted terminal repeat (ITR). The AAV particle and/or its component capsid and viral genome may be engineered to alter tropism to a particular cell-type, tissue, organ or organism.

As used herein, “viral genome” or “vector genome” refers to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

As used herein, a “payload region” is any nucleic acid molecule which encodes one or more “payloads” of the disclosure. As non-limiting examples, a payload region may be a nucleic acid sequence encoding a payload comprising an RNAi agent or a polypeptide.

As used herein, a “targeting peptide” refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein. As a non-limiting example, the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism.

The AAV particles and payloads of the disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In a preferred embodiment, the AAV particles of the disclosure demonstrate enhanced tropism for a target cell type, tissue or organ. As a non-limiting example, the AAV particle may have enhanced tropism for cells and tissues of the central or peripheral nervous systems (CNS and PNS, respectively). The AAV particles of the disclosure may, in addition, or alternatively, have decreased tropism for an undesired target cell-type, tissue or organ.

Adeno-associated viruses (AAV) are small non-enveloped icosahedral capsid viruses of the Parvoviridae family characterized by a single stranded DNA viral genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. The Parvoviridae family comprises the Dependovirus genus which includes AAV, capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

The parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996), the contents of which are incorporated by reference in their entirety.

AAV have proven to be useful as a biological tool due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integration into the host genome and without replicating, and their relatively benign immunogenic profile. The genome of the virus may be manipulated to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to target a particular tissue and express or deliver a desired payload.

The wild-type AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) traditionally cap the viral genome at both the 5′ and the 3′ end, providing origins of replication for the viral genome. While not wishing to be bound by theory, an AAV viral genome typically comprises two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by a self-complementary region (145nt in wild-type AAV) at the 5′ and 3′ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

The wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for the four non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by Rep genes) and one for the three capsid, or structural, proteins (VP1, VP2, VP3, encoded by capsid genes or Cap genes). The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins from a single open reading frame and the generation of three capsid proteins from a single open reading frame. Though it varies by AAV serotype, as a non-limiting example, for AAV9/hu.14 (SEQ ID NO: 123 of U.S. Pat. No. 7,906,111, the contents of which are herein incorporated by reference in their entirety) VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. As a result, changes in the sequence in the VP3 region, are also changes to VP1 and VP2, however, the percent difference as compared to the parent sequence will be greatest for VP3 since it is the shortest sequence of the three. Though described here in relation to the amino acid sequence, the nucleic acid sequence encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1:1:10 of VP1:VP2:VP3. As used herein, an “AAV serotype” is defined primarily by the AAV capsid. In some instances, the ITRs are also specifically described by the AAV serotype (e.g., AAV2/9).

AAV vectors of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, a “vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.

In addition to single stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) viral genomes. scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the transduced cell.

In one embodiment, the AAV particle of the present disclosure is an scAAV.

In one embodiment, the AAV particle of the present disclosure is an ssAAV.

Methods for producing and/or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO2005005610; and WO2005072364, the content of each of which is incorporated herein by reference in its entirety).

In one embodiment, the AAV particles of the disclosure comprising a capsid with an inserted targeting peptide and a viral genome, may have enhanced tropism for a cell-type or tissue of the human CNS.

AAV Capsids

AAV particles of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in characteristics such as, but not limited to, packaging, tropism, transduction and immunogenic profiles. While not wishing to be bound by theory, the AAV capsid protein is often considered to be the driver of AAV particle tropism to a particular tissue.

In one embodiment, an AAV particle may have a capsid protein and ITR sequences derived from the same parent serotype (e.g., AAV2 capsid and AAV2 ITRs). In another embodiment, the AAV particle may be a pseudo-typed AAV particle, wherein the capsid protein and ITR sequences are derived from different parent serotypes (e.g., AAV9 capsid and AAV2 ITRs; AAV2/9).

The AAV particles of the present disclosure may comprise an AAV capsid protein with a targeting peptide inserted into the parent sequence. The parent capsid or serotype may comprise or be derived from any natural or recombinant AAV serotype. As used herein, a “parent” sequence is a nucleotide or amino acid sequence into which a targeting sequence is inserted (i.e., nucleotide insertion into nucleic acid sequence or amino acid sequence insertion into amino acid sequence).

In a preferred embodiment, the parent AAV capsid nucleotide sequence is as set forth in SEQ ID NO: 1.

In another embodiment, the parent AAV capsid nucleotide sequence is a K449R variant of SEQ ID NO: 1, wherein the codon encoding a lysine (e.g., AAA or AAG) at position 449 in the amino acid sequence (nucleotides 1345-1347) is exchanged for one encoding an arginine (CGT, CGC, CGA, CGG, AGA, AGG). The K449R variant has the same function as wild-type AAV9.

In one embodiment, the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 2.

In another embodiment, the parent AAV capsid amino acid sequence is as set forth in SEQ ID NO: 3.

In one embodiment the parent AAV capsid sequence is any of those shown in Table 1.

TABLE 1 AAV Capsid Sequences SEQ Serotype ID NO Reference Information AAV9/hu.14 (nt) 1 U.S. Pat. No. 7,906,111 SEQ ID NO: 3; WO2015038958 SEQ ID NO: 11 AAV9/hu.14 (aa) 2 U.S. Pat. No. 7,906,111 SEQ ID NO: 123; WO2015038958 SEQ ID NO: 2 AAV9/hu.14 K449R (aa) 3 WO2017100671 SEQ ID NO: 45

Each of the patents, applications and or publications listed in Table 1 are hereby incorporated by reference in their entirety.

The parent AAV serotype and associated capsid sequence may be any of those known in the art. Non-limiting examples of such AAV serotypes include, AAV9, AAV9 K449R (or K449R AAV9), AAV1, AAVrh10, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAVS-3/rh.57, AAVS-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.10, AAV16.12/hu.11, AAV29.3/bb .1, AAV29.5/bb .2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33 .4/hu. 15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVCS, AAVF3, AAVFS, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, and/or AAVF9/HSC9 and variants thereof.

In some embodiments, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008), US Publication US20140359799 and U.S. Pat. No. 7,588,772, each of which is herein incorporated by reference in its entirety). The amino acid sequence of AAVDJ8 may comprise two or more mutations in order to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence is as described by SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, the contents of which are herein incorporated by reference in their entirety, and the AAVDJ8 sequence may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, the AAVDJ8 sequence may comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590T where arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

In one embodiment, the parent AAV capsid sequence comprises an AAV9 sequence.

In one embodiment, the parent AAV capsid sequence comprises an K449R AAV9 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVDJ sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVDJ8 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAVrh10 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAV1 sequence.

In one embodiment, the parent AAV capsid sequence comprises an AAV5 sequence.

While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In one embodiment, a parent AAV capsid sequence comprises a VP1, VP2 and/or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e. capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the peptide sequence (Met1), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Met1) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met/AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins.

Where the Met/AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include a Met1/AA1 amino acid (Met+/AA+) and some of which may lack a Met1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−). For further discussion regarding Met/AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 October 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 Feb. 19. 327(5968): 973-977; the contents of which are each incorporated herein by reference in its entirety.

According to the present disclosure, references to capsid proteins is not limited to either clipped (Met−/AA−) or unclipped (Met+/AA+) and may, in context, refer to independent capsid proteins, viral capsids comprised of a mixture of capsid proteins, and/or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid proteins of the present disclosure. A direct reference to a “capsid protein” or “capsid polypeptide” (such as VP1, VP2 or VP2) may also comprise VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) as well as corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA-clipping (Met−/AA−).

Further according to the present disclosure, a reference to a specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid proteins which include a Met1/AA1 amino acid (Met+/AA+) should be understood to teach the VP capsid proteins which lack the Met1/AA1 amino acid as upon review of the sequence, it is readily apparent any sequence which merely lacks the first listed amino acid (whether or not Met1/AA1).

As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Met1” amino acid (Met+) encoded by the AUG/ATG start codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Met1” amino acid (Met−) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon may also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1−) of the 736 amino acid AA1+sequence.

References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Met1/AA1 amino acid (Met+/AA1+), corresponding VP capsid proteins which lack the Met1/AA1 amino acid as a result of Met/AA1-clipping (Met−/AA1−), and combinations thereof (Met+/AA1+ and Met−/AA1−).

As a non-limiting example, an AAV capsid serotype can include VP1 (Met+/AA1+), VP1 (Met−/AA1−), or a combination of VP1 (Met+/AA1+) and VP1 (Met−/AA1−). An AAV capsid serotype can also include VP3 (Met+/AA1+), VP3 (Met−/AA1−), or a combination of VP3 (Met+/AA1+) and VP3 (Met−/AA1−); and can also include similar optional combinations of VP2 (Met+/AA1) and VP2 (Met−/AA1−).

In one embodiment, the parent AAV capsid sequence may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the those described above.

In one embodiment, the parent AAV capsid sequence may be encoded by a nucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of those described above.

In one embodiment, the parent sequence is not an AAV capsid sequence and is instead a different vector (e.g., lentivirus, plasmid, etc.). In another embodiment, the parent sequence is a delivery vehicle (e.g., a nanoparticle) and the targeting peptide is attached thereto.

Targeting Peptides

Disclosed herein are targeting peptides and associated AAV particles comprising a capsid protein with one or more targeting peptide inserts, for enhanced or improved transduction of a target tissue (e.g., cells of the CNS or PNS).

In one embodiment, the targeting peptide may direct an AAV particle to a cell or tissue of the CNS. The cell of the CNS may be, but is not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and/or supporting cells of the brain such as immune cells (e.g., T cells). The tissue of the CNS may be, but is not limited to, the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei.

In one embodiment, the targeting peptide may direct an AAV particle to a cell or tissue of the PNS. The cell or tissue of the PNS may be, but is not limited to, a dorsal root ganglion (DRG).

The targeting peptide may direct an AAV particle to the CNS (e.g., the cortex) after intravenous administration.

The targeting peptide may direct and AAV particle to the PNS (e.g., DRG) after intravenous administration.

A targeting peptide may vary in length. In one embodiment, the targeting peptide is 3-20 amino acids in length. As non-limiting examples, the targeting peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length.

Targeting peptides of the present disclosure may be identified and/or designed by any method known in the art. As a non-limiting example, the CREATE system as described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and in International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are herein incorporated by reference in their entirety, may be used as a means of identifying targeting peptides, in either mice or other research animals, such as, but not limited to, non-human primates.

Targeting peptides and associated AAV particles may be identified from libraries of AAV capsids comprised of targeting peptide variants. In one embodiment, the targeting peptides may be 7 amino acid sequences (7-mers). In another embodiment, the targeting peptides may be 9 amino acid sequences (9-mers). The targeting peptides may also differ in their method of creation or design, with non-limiting examples including, random peptide selection, site saturation mutagenesis, and/or optimization of a particular region of the peptide (e.g., flanking regions or central core).

In one embodiment, a targeting peptide library comprises targeting peptides of 7 amino acids (7-mer) in length randomly generated by PCR.

In one embodiment, a targeting peptide library comprises targeting peptides with 3 mutated amino acids. In one embodiment, these 3 mutated amino acids are consecutive amino acids. In another embodiment, these 3 mutated amino acids are not consecutive amino acids. In one embodiment, the parent targeting peptide is a 7-mer. In another embodiment, the parent peptide is a 9-mer.

In one embodiment, a targeting peptide library comprises 7-mer targeting peptides, wherein the amino acids of the targeting peptide and/or the flanking sequences are evolved through site saturation mutagenesis of 3 consecutive amino acids. In one embodiment, NNK (N=any base; K=G or T) codons are used to generate the site saturated mutation sequences.

AAV particles comprising capsid proteins with targeting peptide inserts are generated and viral genomes encoding a reporter (e.g., GFP) encapsulated within. These AAV particles (or AAV capsid library) are then administered to a transgenic mouse by intravenous delivery to the tail vein. Administration of these capsid libraries to cre-expressing mice results in expression of the reporter payload in the target tissue, due to the expression of Cre.

AAV particles and/or viral genomes may be recovered from the target tissue for identification of targeting peptides and associated AAV particles that are enriched, indicating enhanced transduction of target tissue. Standard methods in the art, such as, but not limited to next generation sequencing (NGS), viral genome quantification, biochemical assays, immunohistochemistry and/or imaging of target tissue samples may be used to determine enrichment.

A target tissue may be any cell, tissue or organ of a subject. As non-limiting examples, samples may be collected from brain, spinal cord, dorsal root ganglia and associated roots, liver, heart, gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal glands, stomach, sciatic nerve, saphenous nerve, thyroid gland, eyes (with or without optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, skin of the leg, superior cervical ganglia, urinary bladder, ovaries, uterus, prostate gland, testes, and/or any sites identified as having a lesion, or being of interest.

Targeting Peptide Sequences

In one embodiment the targeting peptide may comprise a sequence as set forth in Table 2. In Table 2, “_1” refers to NNM codons where A or C is in the third position and “_2” refers to NNK codons where G or T is in the third position. Additionally, the NNM codons cannot cover the entire repertoire of amino acids since Met or Trp can only be encoded by codons ATG and TGG, respectively. Therefore, some “NNM” sequences also contain some codons ending in G.

TABLE 2 Peptides Peptide SEQ Peptide SEQ Sequence_ID ID NO: Sequence_ID ID NO: AQAGAGSER_1 194 DGTGQVTGW_1  68 AQAGAGSER_2 194 DGTGQVTGW_2  68 AQDQNPGRW_1 195 DGTGRLTGW_1 159 AQDQNPGRW_2 195 DGTGRLTGW_2 159 AQELTRPFL_1 144 DGTGRTVGW_1 117 AQELTRPFL_2 144 DGTGRTVGW_2 117 AQEVPGYRW_1 196 DGTGSGMMT_1 306 AQEVPGYRW_2 196 DGTGSGMMT_2 306 AQFPTNYDS_1  66 DGTGSISGW_1 307 AQFPTNYDS_2  66 DGTGSISGW_2 307 AQFVVGQQY_1  95 DGTGSLAGW_1 308 AQFVVGQQY_2  95 DGTGSLAGW_2 308 AQGASPGRW_1 149 DGTGSLNGW_1 309 AQGASPGRW_2 149 DGTGSLNGW_2 309 AQGENPGRW_1  96 DGTGSLQGW_1 310 AQGENPGRW_2  96 DGTGSLQGW_2 310 AQGGNPGRW_1  91 DGTGSLSGW_1 311 AQGGNPGRW_2  91 DGTGSLSGW_2 311 AQGGSTGSN_1 197 DGTGSLVGW_1 312 AQGGSTGSN_2 197 DGTGSLVGW_2 312 AQGPTRPFL_1 125 DGTGSTHGW_1 119 AQGPTRPFL_2 125 DGTGSTHGW_2 119 AQGRDGWAA_1 198 DGTGSTKGW_1 313 AQGRDGWAA_2 198 DGTGSTKGW_2 313 AQGRMTDSQ_1 199 DGTGSTMGW_1 314 AQGRMTDSQ_2 199 DGTGSTMGW_2 314 AQGSDVGRW_1 128 DGTGSTQGW_1 315 AQGSDVGRW_2 128 DGTGSTQGW_2 315 AQGSNPGRW_1 103 DGTGSTSGW_1 316 AQGSNPGRW_2 103 DGTGSTSGW_2 316 AQGSNSPQV_1 200 DGTGSTTGW_1 134 AQGSNSPQV_2 200 DGTGSTTGW_2 134 AQGSWNPPA_1  80 DGTGSVMGW_1 317 AQGSWNPPA_2  80 DGTGSVMGW_2 317 AQGTWNPPA_1  82 DGTGSVTGW_1 318 AQGTWNPPA_2  82 DGTGSVTGW_2 318 AQGVFIPPK_1 201 DGTGTLAGW_1 319 AQGVFIPPK_2 201 DGTGTLAGW_2 319 AQHVNASQS_1 202 DGTGTLHGW_1 320 AQHVNASQS_2 202 DGTGTLHGW_2 320 AQIKAGWAQ_1 203 DGTGTLKGW_1 321 AQIKAGWAQ_2 203 DGTGTLKGW_2 321 AQIMSGYAQ_1 204 DGTGTLSGW_1 322 AQIMSGYAQ_2 204 DGTGTLSGW_2 322 AQKSVGSVY_1 205 DGTGTTLGW_1 323 AQKSVGSVY_2 205 DGTGTTLGW_2 323 AQLEHGFAQ_1 206 DGTGTTMGW_1 324 AQLEHGFAQ_2 206 DGTGTTMGW_2 324 AQLGGVLSA_1 207 DGTGTTTGW_1 130 AQLGGVLSA_2 207 DGTGTTTGW_2 130 AQLGLSQGR_1 208 DGTGTTVGW_1  74 AQLGLSQGR_2 208 DGTGTTVGW_2  74 AQLGYGFAQ_1 209 DGTGTTYGW_1 325 AQLGYGFAQ_2 209 DGTGTTYGW_2 325 AQLKYGLAQ_1 115 DGTGTVHGW_1 326 AQLKYGLAQ_2 115 DGTGTVHGW_2 326 AQLRIGFAQ_1 210 DGTGTVQGW_1 327 AQLRIGFAQ_2 210 DGTGTVQGW_2 327 AQLRMGYSQ_1 211 DGTGTVSGW_1 328 AQLRMGYSQ_2 211 DGTGTVSGW_2 328 AQLRQGYAQ_1 212 DGTGTVTGW_1 329 AQLRQGYAQ_2 212 DGTGTVTGW_2 329 AQLRVGFAQ_1 123 DGTHARLSS_1 330 AQLRVGFAQ_2 123 DGTHARLSS_2 330 AQLSCRSQM_1 213 DGTHAYMAS_1 153 AQLSCRSQM_2 213 DGTHAYMAS_2 153 AQLTYSQSL_1 214 DGTHFAPPR_1 112 AQLTYSQSL_2 214 DGTHFAPPR_2 112 AQLYKGYSQ_1 215 DGTHIHLSS_1 162 AQLYKGYSQ_2 215 DGTHIHLSS_2 162 AQMPQRPFL_1 216 DGTHIRALS_1 331 AQMPQRPFL_2 216 DGTHIRALS_2 331 AQNGNPGRW_1  84 DGTHIRLAS_1 332 AQNGNPGRW_2  84 DGTHIRLAS_2 332 AQPEGSARW_1  60 DGTHLQPFR_1 333 AQPEGSARW_2  60 DGTHLQPFR_2 333 AQPLAVYGA_1 217 DGTHSFYDA_1 334 AQPLAVYGA_2 217 DGTHSFYDA_2 334 AQPQSSSMS_1 218 DGTHSTTGW_1 145 AQPQSSSMS_2 218 DGTHSTTGW_2 145 AQPSVGGYW_1 219 DGTHTRTGW_1  90 AQPSVGGYW_2 219 DGTHTRTGW_2  90 AQQAVGQSW_1 220 DGTHVRALS_1 335 AQQAVGQSW_2 220 DGTHVRALS_2 335 AQQRSLASG_1 221 DGTHVYMAS_1 336 AQQRSLASG_2 221 DGTHVYMAS_2 336 AQQVMNSQG_1 222 DGTHVYMSS_1 337 AQQVMNSQG_2 222 DGTHVYMSS_2 337 AQRGVGLSQ_1 223 DGTIALPFK_1 338 AQRGVGLSQ_2 223 DGTIALPFK_2 338 AQRHDAEGS_1 224 DGTIALPFR_1 339 AQRHDAEGS_2 224 DGTIALPFR_2 339 AQRKGEPHY_1 225 DGTIATRYV_1 340 AQRKGEPHY_2 225 DGTIATRYV_2 340 AQRYTGDSS_1 138 DGTIERPFR_1  87 AQRYTGDSS_2 138 DGTIERPFR_2  87 AQSAMAAKG_1 226 DGTIGYAYV_1 341 AQSAMAAKG_2 226 DGTIGYAYV_2 341 AQSGGLTGS_1 227 DGTIQAPFK_1 342 AQSGGLTGS_2 227 DGTIQAPFK_2 342 AQSGGVGQV_1 228 DGTIRLPFK_1 343 AQSGGVGQV_2 228 DGTIRLPFK_2 343 AQSLATPFR_1 169 DGTISKEVG_1 344 AQSLATPFR_2 169 DGTISKEVG_2 344 AQSMSRPFL_1 229 DGTISQPFK_1 105 AQSMSRPFL_2 229 DGTISQPFK_2 105 AQSQLRPFL_1 230 DGTKIQLSS_1 146 AQSQLRPFL_2 230 DGTKIQLSS_2 146 AQSVAKPFL_1 231 DGTKIRLSS_1 111 AQSVAKPFL_2 231 DGTKIRLSS_2 111 AQSVSQPFR_1 232 DGTKLMLSS_1 157 AQSVSQPFR_2 232 DGTKLMLSS_2 157 AQSVVRPFL_1 233 DGTKLRLSS_1 118 AQSVVRPFL_2 233 DGTKLRLSS_2 118 AQTALSSST_1 234 DGTKMVLQL_1 142 AQTALSSST_2 234 DGTKMVLQL_2 142 AQTEMGGRC_1 235 DGTKSLVQL_1 345 AQTEMGGRC_2 235 DGTKSLVQL_2 345 AQTGFAPPR_1 161 DGTKVLVQL_1 122 AQTGFAPPR_2 161 DGTKVLVQL_2 122 AQTIRGYSS_1 236 DGTLAAPFK_1 120 AQTIRGYSS_2 236 DGTLAAPFK_2 120 AQTISNYHT_1 237 DGTLAVNFK_1 346 AQTISNYHT_2 237 DGTLAVNFK_2 346 AQTLARPFV_1  98 DGTLAVPFK_1  71 AQTLARPFV_2  98 DGTLAVPFK_2  71 AQTLAVPFK_1 168 DGTLAYPFK_1 347 AQTLAVPFK_2 168 DGTLAYPFK_2 347 AQTPDRPWL_1 238 DGTLERPFR_1 156 AQTPDRPWL_2 238 DGTLERPFR_2 156 AQTRAGYAQ_1 126 DGTLEVHFK_1 348 AQTRAGYAQ_2 126 DGTLEVHFK_2 348 AQTRAGYSQ_1 141 DGTLLRLSS_1 121 AQTRAGYSQ_2 141 DGTLLRLSS_2 121 AQTREYLLG_1  93 DGTLNNPFR_1 109 AQTREYLLG_2  93 DGTLNNPFR_2 109 AQTSAKPFL_1 163 DGTLQQPFR_1  89 AQTSAKPFL_2 163 DGTLQQPFR_2  89 AQTSARPFL_1 100 DGTLSQPFR_1  65 AQTSARPFL_2 100 DGTLSQPFR_2  65 AQTTDRPFL_1  85 DGTLSRTLW_1 349 AQTTDRPFL_2  85 DGTLSRTLW_2 349 AQTTEKPWL_1  83 DGTLSSPFR_1 350 AQTTEKPWL_2  83 DGTLSSPFR_2 350 AQTVARPFY_1 239 DGTLTVPFR_1 351 AQTVARPFY_2 239 DGTLTVPFR_2 351 AQTVATPFR_1 240 DGTLVAPFR_1 352 AQTVATPFR_2 240 DGTLVAPFR_2 352 AQTVTQLFK_1 241 DGTMDKPFR_1  70 AQTVTQLFK_2 241 DGTMDKPFR_2  70 AQVHVGSVY_1 165 DGTMDRPFK_1 102 AQVHVGSVY_2 165 DGTMDRPFK_2 102 AQVLAGYNM_1 242 DGTMLRLSS_1 148 AQVLAGYNM_2 242 DGTMLRLSS_2 148 AQVSEARVR_1 243 DGTMQLTGW_1 353 AQVSEARVR_2 243 DGTMQLTGW_2 353 AQVVVGYSQ_1 244 DGTNGLKGW_1  76 AQVVVGYSQ_2 244 DGTNGLKGW_2  76 AQWAAGYNV_1 245 DGTNSISGW_1 354 AQWAAGYNV_2 245 DGTNSISGW_2 354 AQWELSNGY_1 246 DGTNSLSGW_1 355 AQWELSNGY_2 246 DGTNSLSGW_2 355 AQWEVKGGY_1 247 DGTNSTTGW_1 143 AQWEVKGGY_2 247 DGTNSTTGW_2 143 AQWEVKRGY_1 248 DGTNSVTGW_1 356 AQWEVKRGY_2 248 DGTNSVTGW_2 356 AQWEVQSGF_1 249 DGTNTINGW_1 124 AQWEVQSGF_2 249 DGTNTINGW_2 124 AQWEVRGGY_1 250 DGTNTLGGW_1 357 AQWEVRGGY_2 250 DGTNTLGGW_2 357 AQWEVTSGW_1 251 DGTNTTHGW_1 113 AQWEVTSGW_2 251 DGTNTTHGW_2 113 AQWGAPSHG_1 252 DGTNYRLSS_1 358 AQWGAPSHG_2 252 DGTNYRLSS_2 358 AQWMELGSS_1 253 DGTQALSGW_1 359 AQWMELGSS_2 253 DGTQALSGW_2 359 AQWMFGGSG_1 254 DGTQFRLSS_1 129 AQWMFGGSG_2 254 DGTQFRLSS_2 129 AQWMLGGAQ_1 255 DGTQFSPPR_1 108 AQWMLGGAQ_2 255 DGTQFSPPR_2 108 AQWPTAYDA_1 256 DGTQGLKGW_1 158 AQWPTAYDA_2 256 DGTQGLKGW_2 158 AQWPTSYDA_1  62 DGTQTTSGW_1 360 AQWPTSYDA_2  62 DGTQTTSGW_2 360 AQWQVQTGF_1 257 DGTRALTGW_1 361 AQWQVQTGF_2 257 DGTRALTGW_2 361 AQWSTEGGY_1 258 DGTRFSLSS_1 362 AQWSTEGGY_2 258 DGTRFSLSS_2 362 AQWTAAGGY_1 259 DGTRGLSGW_1 363 AQWTAAGGY_2 259 DGTRGLSGW_2 363 AQWTTESGY_1 260 DGTRIGLSS_1 364 AQWTTESGY_2 260 DGTRIGLSS_2 364 AQWVYGSSH_1 261 DGTRLHLAS_1 365 AQWVYGSSH_2 261 DGTRLHLAS_2 365 AQYLAGYTV_1 262 DGTRLHLSS_1 366 AQYLAGYTV_2 262 DGTRLHLSS_2 366 AQYLKGYSV_1 152 DGTRLLLSS_1 367 AQYLKGYSV_2 152 DGTRLLLSS_2 367 AQYLSGYNT_1 263 DGTRLMLSS_1 368 AQYLSGYNT_2 263 DGTRLMLSS_2 368 DGAAATTGW_1 264 DGTRLNLSS_1 369 DGAAATTGW_2 264 DGTRLNLSS_2 369 DGAGGTSGW_1 151 DGTRMVVQL_1 370 DGAGGTSGW_2 151 DGTRMVVQL_2 370 DGAGTTSGW_1 265 DGTRNMYEG_1 135 DGAGTTSGW_2 265 DGTRNMYEG_2 135 DGAHGLSGW_1 266 DGTRSITGW_1 371 DGAHGLSGW_2 266 DGTRSITGW_2 371 DGAHVGLSS_1 267 DGTRSLHGW_1 372 DGAHVGLSS_2 267 DGTRSLHGW_2 372 DGARTVLQL_1 268 DGTRSTTGW_1 373 DGARTVLQL_2 268 DGTRSTTGW_2 373 DGEYQKPFR_1 269 DGTRTTTGW_1 106 DGEYQKPFR_2 269 DGTRTTTGW_2 106 DGGGTTTGW_1 270 DGTRTVTGW_1 374 DGGGTTTGW_2 270 DGTRTVTGW_2 374 DGHATSMGW_1 271 DGTRTVVQL_1 375 DGHATSMGW_2 271 DGTRTVVQL_2 375 DGKGSTQGW_1 272 DGTRVHLSS_1 376 DGKGSTQGW_2 272 DGTRVHLSS_2 376 DGKQYQLSS_1  92 DGTSFPYAR_1  86 DGKQYQLSS_2  92 DGTSFPYAR_2  86 DGNGGLKGW_1 167 DGTSFTPPK_1  81 DGNGGLKGW_2 167 DGTSFTPPK_2  81 DGQGGLSGW_1 273 DGTSFTPPR_1  88 DGQGGLSGW_2 273 DGTSFTPPR_2  88 DGQHFAPPR_1 110 DGTSGLHGW_1 377 DGQHFAPPR_2 110 DGTSGLHGW_2 377 DGRATKTLY_1 274 DGTSGLKGW_1 101 DGRATKTLY_2 274 DGTSGLKGW_2 101 DGRNALTGW_1 275 DGTSIHLSS_1 378 DGRNALTGW_2 275 DGTSIHLSS_2 378 DGRRQVIQL_1 276 DGTSIMLSS_1 379 DGRRQVIQL_2 276 DGTSIMLSS_2 379 DGRVYGLSS_1 277 DGTSLRLSS_1 166 DGRVYGLSS_2 277 DGTSLRLSS_2 166 DGSGRTTGW_1 147 DGTSNYGAR_1 380 DGSGRTTGW_2 147 DGTSNYGAR_2 380 DGSGTTRGW_1 114 DGTSSYYDA_1 381 DGSGTTRGW_2 114 DGTSSYYDA_2 381 DGSGTVSGW_1 278 DGTSSYYDS_1  59 DGSGTVSGW_2 278 DGTSSYYDS_2  59 DGSPEKPFR_1 160 DGTSTISGW_1 382 DGSPEKPFR_2 160 DGTSTISGW_2 382 DGSQSTTGW_1 136 DGTSTITGW_1 383 DGSQSTTGW_2 136 DGTSTITGW_2 383 DGSSFYPPK_1 127 DGTSTLHGW_1 384 DGSSFYPPK_2 127 DGTSTLHGW_2 384 DGSSSYYDA_1  64 DGTSTLRGW_1 385 DGSSSYYDA_2  64 DGTSTLRGW_2 385 DGSIERPFR_1  99 DGTSTLSGW_1 386 DGSIERPFR_2  99 DGTSTLSGW_2 386 DGTAARLSS_1 132 DGTSYVPPK_1  97 DGTAARLSS_2 132 DGTSYVPPK_2  97 DGTADKPFR_1  63 DGTSYVPPR_1  78 DGTADKPFR_2  63 DGTSYVPPR_2  78 DGTADRPFR_1 155 DGTTATYYK_1 387 DGTADRPFR_2 155 DGTTATYYK_2 387 DGTAERPFR_1 140 DGTTFTPPR_1  79 DGTAERPFR_2 140 DGTTFTPPR_2  79 DGTAIHLSS_1  67 DGTTLAPFR_1 388 DGTAIHLSS_2  67 DGTTLAPFR_2 388 DGTAIYLSS_1 279 DGTTLVPPR_1 116 DGTAIYLSS_2 279 DGTTLVPPR_2 116 DGTALMLSS_1 280 DGTTSKTLW_1 389 DGTALMLSS_2 280 DGTTSKTLW_2 389 DGTASISGW_1 281 DGTTSRTLW_1 390 DGTASISGW_2 281 DGTTSRTLW_2 390 DGTASTSGW_1 282 DGTTTRSLY_1 391 DGTASTSGW_2 282 DGTTTRSLY_2 391 DGTASVTGW_1 283 DGTTTTTGW_1 392 DGTASVTGW_2 283 DGTTTTTGW_2 392 DGTASYYDS_1  61 DGTTTYGAR_1  77 DGTASYYDS_2  61 DGTTTYGAR_2  77 DGTATTMGW_1 284 DGTTWTPPR_1 139 DGTATTMGW_2 284 DGTTWTPPR_2 139 DGTATTTGW_1 285 DGTTYMLSS_1 393 DGTATTTGW_2 285 DGTTYMLSS_2 393 DGTAYRLSS_1 286 DGTTYVPPR_1  75 DGTAYRLSS_2 286 DGTTYVPPR_2  75 DGTDKMWSL_1 287 DGTVANPFR_1 394 DGTDKMWSL_2 287 DGTVANPFR_2 394 DGTGGIKGW_1 131 DGTVDRPFK_1 395 DGTGGIKGW_2 131 DGTVDRPFK_2 395 DGTGGIMGW_1 288 DGTVIHLSS_1  73 DGTGGIMGW_2 288 DGTVIHLSS_2  73 DGTGGISGW_1 289 DGTVILLSS_1 396 DGTGGISGW_2 289 DGTVILLSS_2 396 DGTGGLAGW_1 290 DGTVIMLSS_1 397 DGTGGLAGW_2 290 DGTVIMLSS_2 397 DGTGGLHGW_1 291 DGTVLHLSS_1 398 DGTGGLHGW_2 291 DGTVLHLSS_2 398 DGTGGLQGW_1 292 DGTVLMLSS_1 399 DGTGGLQGW_2 292 DGTVLMLSS_2 399 DGTGGLRGW_1 154 DGTVLVPFR_1 150 DGTGGLRGW_2 154 DGTVLVPFR_2 150 DGTGGLSGW_1 293 DGTVPYLAS_1 400 DGTGGLSGW_2 293 DGTVPYLAS_2 400 DGTGGLTGW_1 294 DGTVPYLSS_1 401 DGTGGLTGW_2 294 DGTVPYLSS_2 401 DGTGGTKGW_1 107 DGTVRVPFR_1 164 DGTGGTKGW_2 107 DGTVRVPFR_2 164 DGTGGTSGW_1 295 DGTVSMPFK_1 402 DGTGGTSGW_2 295 DGTVSMPFK_2 402 DGTGGVHGW_1 296 DGTVSNPFR_1 403 DGTGGVHGW_2 296 DGTVSNPFR_2 403 DGTGGVMGW_1 297 DGTVSTRWV_1 404 DGTGGVMGW_2 297 DGTVSTRWV_2 404 DGTGGVSGW_1 298 DGTVTTTGW_1 405 DGTGGVSGW_2 298 DGTVTTTGW_2 405 DGTGGVTGW_1 299 DGTVTVTGW_1 406 DGTGGVTGW_2 299 DGTVTVTGW_2 406 DGTGGVYGW_1 300 DGTVWVPPR_1 407 DGTGGVYGW_2 300 DGTVWVPPR_2 407 DGTGNLQGW_1 301 DGTVYRLSS_1 408 DGTGNLQGW_2 301 DGTVYRLSS_2 408 DGTGNLRGW_1 133 DGTYARLSS_1 409 DGTGNLRGW_2 133 DGTYARLSS_2 409 DGTGNLSGW_1 302 DGTYGNKLW_1 410 DGTGNLSGW_2 302 DGTYGNKLW_2 410 DGTGNTHGW_1  72 DGTYIHLSS_1 411 DGTGNTHGW_2  72 DGTYIHLSS_2 411 DGTGNTRGW_1  94 DGTYSTSGW_1 412 DGTGNTRGW_2  94 DGTYSTSGW_2 412 DGTGNTSGW_1 137 DGVHPGLSS_1 104 DGTGNTSGW_2 137 DGVHPGLSS_2 104 DGTGNVSGW_1 303 DGVVALLAS_1 413 DGTGNVSGW_2 303 DGVVALLAS_2 413 DGTGNVTGW_1  69 DGYVGVGSL_1 414 DGTGNVTGW_2  69 DGYVGVGSL_2 414 DGTGQLVGW_1 304 control (wtAAV9- NNM) DGTGQLVGW_2 304 control (wtAAV9- NNK) DGTGQTIGW_1 305 DGTGQTIGW_2 305

In one embodiment, the targeting peptide may comprise an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences shown in Table 2.

In one embodiment, a targeting peptide may comprise 4 or more contiguous amino acids of any of the targeting peptides disclosed herein. In one embodiment the targeting peptide may comprise 4 contiguous amino acids of any of the sequences as set forth in Table 2. In one embodiment the targeting peptide may comprise 5 contiguous amino acids of any of the sequences as set forth in Table 2. In one embodiment the targeting peptide may comprise 6 contiguous amino acids of any of the sequences as set forth in Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence comprising at least 4 contiguous amino acids of any of the sequences as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid with a targeting peptide insert, wherein the targeting peptide has an amino acid sequence substantially comprising any of the sequences as set forth in any of Table 2.

In one embodiment, the AAV particle of the disclosure comprises an AAV capsid polynucleotide with a targeting nucleic acid insert, wherein the targeting nucleic acid insert has a nucleotide sequence substantially comprising any of those set forth as Table 2.

The AAV particle of the disclosure comprising a targeting nucleic acid insert, may have a polynucleotide sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence.

The AAV particle of the disclosure comprising a targeting peptide insert, may have an amino acid sequence with 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, identity to the parent capsid sequence.

In any of the DNA and RNA sequences referenced and/or described herein, the single letter symbol has the following description: A for adenine; C for cytosine; G for guanine; T for thymine; U for Uracil; W for weak bases such as adenine or thymine; S for strong nucleotides such as cytosine and guanine; M for amino nucleotides such as adenine and cytosine; K for keto nucleotides such as guanine and thymine; R for purines adenine and guanine; Y for pyrimidine cytosine and thymine; B for any base that is not A (e.g., cytosine, guanine, and thymine); D for any base that is not C (e.g., adenine, guanine, and thymine); H for any base that is not G (e.g., adenine, cytosine, and thymine); V for any base that is not T (e.g., adenine, cytosine, and guanine); N for any nucleotide (which is not a gap); and Z is for zero.

In any of the amino acid sequences referenced and/or described herein, the single letter symbol has the following description: G (Gly) for Glycine; A (Ala) for Alanine; L (Leu) for Leucine; M (Met) for Methionine; F (Phe) for Phenylalanine; W (Trp) for Tryptophan; K (Lys) for Lysine; Q (Gln) for Glutamine; E (Glu) for Glutamic Acid; S (Ser) for Serine; P (Pro) for Proline; V (Val) for Valine; I (Ile) for Isoleucine; C (Cys) for Cysteine; Y (Tyr) for Tyrosine; H (His) for Histidine; R (Arg) for Arginine; N (Asn) for Asparagine; D (Asp) for Aspartic Acid; T (Thr) for Threonine; B (Asx) for Aspartic acid or Asparagine; J (Xle) for Leucine or Isoleucine; O (Pyl) for Pyrrolysine; U (Sec) for Selenocysteine; X (Xaa) for any amino acid; and Z (Glx) for Glutamine or Glutamic acid.

Use of Targeting Peptides in AAV Particles

Targeting peptides may be stand-alone peptides or may be inserted into or conjugated to a parent sequence. In one embodiment, the targeting peptides are inserted into the capsid protein of an AAV particle.

One or more targeting peptides may be inserted into a parent AAV capsid sequence to generate the AAV particles of the disclosure.

Targeting peptides may be inserted into a parent AAV capsid sequence in any location that results in fully functional AAV particles. The targeting peptide may be inserted in VP1, VP2 and/or VP3. Numbering of the amino acid residues differs across AAV serotypes, and so the exact amino acid position of the targeting peptide insertion may not be critical. As used herein, amino acid positions of the parent AAV capsid sequence are described using AAV9 (SEQ ID NO: 2) as reference.

In one embodiment, the targeting peptides are inserted in a hypervariable region of the AAV capsid sequence. Non-limiting examples of such hypervariable regions include Loop IV and Loop VIII of the parent AAV capsid. While not wishing to be bound by theory, these surface exposed loops are unstructured and poorly conserved, making them ideal regions for insertion of targeting peptides.

In one embodiment, the targeting peptide is inserted into Loop IV. In another embodiment, the targeting peptide is used to replace a portion, or all of Loop IV. As a non-limiting example, addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.

In one embodiment, the targeting peptide is inserted into Loop VIII. In another embodiment, the targeting peptide is used to replace a portion, or all of Loop VIII. As a non-limiting example, addition of the targeting peptide to the parent AAV capsid sequence may result in the replacement or mutation of at least one amino acid of the parent AAV capsid.

In one embodiment, more than one targeting peptide is inserted into a parent AAV capsid sequence. As a non-limiting example, targeting peptides may be inserted at both Loop IV and Loop VIII in the same parent AAV capsid sequence.

Targeting peptides may be inserted at any amino acid position of the parent AAV capsid sequence, such as, but not limited to, between amino acids at positions 586-592, 588-589, 586-589, 452-458, 262-269, 464-473, 491-495, 546-557 and/or 659-668.

In a preferred embodiment, the targeting peptides are inserted into a parent AAV capsid sequence between amino acids at positions 588 and 589 (Loop VIII). In one embodiment, the parent AAV capsid is AAV9 (SEQ ID NO: 2). In a second embodiment, the parent AAV capsid is K449R AAV9 (SEQ ID NO: 3).

The targeting peptides described herein may increase the transduction of the AAV particles of the disclosure to a target tissue as compared to the parent AAV particle lacking a targeting peptide insert. In one embodiment, the targeting peptide increases the transduction of an AAV particle to a target tissue by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the CNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the PNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

In one embodiment, the targeting peptide increases the transduction of an AAV particle to a cell or tissue of the DRG by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more as compared to a parent AAV particle lacking a targeting peptide insert.

AAV Production

Viral production disclosed herein describes processes and methods for producing AAV particles (with enhanced, improved and/or increased tropism for a target tissue) that may be used to contact a target cell to deliver a payload.

The present disclosure provides methods for the generation of AAV particles comprising targeting peptides. In one embodiment, the AAV particles are prepared by viral genome replication in a viral replication cell. Any method known in the art may be used for the preparation of AAV particles. In one embodiment, AAV particles are produced in mammalian cells (e.g., HEK293). In another embodiment, AAV particles are produced in insect cells (e.g., Sf9)

Methods of making AAV particles are well known in the art and are described in e.g., U.S. Pat. Nos. 6,204,059, 5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526, 7,291,498 and 7,491,508, 5,064,764, 6,194,191, 6,566,118, 8,137,948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, WO2000055342, WO2000075353 and WO2001023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); the contents of each of which are herein incorporated by reference in their entirety. In one embodiment, the AAV particles are made using the methods described in International Patent Publication WO2015191508, the contents of which are herein incorporated by reference in their entirety.

Therapeutic Applications

The present disclosure provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject an AAV particle described herein where the AAV particle comprises the novel capsids (“TRACER AAV particles”) defined by the present disclosure or administering to the subject any of the described compositions, including pharmaceutical compositions, described herein.

In one embodiment, the TRACER AAV particles of the present disclosure are administered to a subject prophylactically, to prevent on-set of disease. In another embodiment, the TRACER AAV particles of the present disclosure are administered to treat (lessen the effects of) a disease or symptoms thereof. In yet another embodiment, the TRACER AAV particles of the present disclosure are administered to cure (eliminate) a disease. In another embodiment, the TRACER AAV particles of the present disclosure are administered to prevent or slow progression of disease. In yet another embodiment, the TRACER AAV particles of the present disclosure are used to reverse the deleterious effects of a disease. Disease status and/or progression may be determined or monitored by standard methods known in the art.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of neurological diseases and/or disorders.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of tauopathy.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Alzheimer's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Friedreich's ataxia, or any disease stemming from a loss or partial loss of frataxin protein.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Parkinson's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Amyotrophic lateral sclerosis.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Huntington's Disease.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of chronic or neuropathic pain.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the central nervous system.

In some embodiments, the TRACER AAV particles of the disclosure are useful in the field of medicine for treatment, prophylaxis, palliation or amelioration of a disease associated with the peripheral nervous system.

In one embodiment, the TRACER AAV particles of the present disclosure are administered to a subject having at least one of the diseases or symptoms described herein.

As used herein, any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons) may be considered a “neurological disease”.

Any neurological disease may be treated with the TRACER AAV particles of the disclosure, or pharmaceutical compositions thereof, including but not limited to, Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS—Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Bechet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbar palsy, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Concentric sclerosis (Balo's sclerosis), Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Chronic progressive external ophtalmoplegia, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia—Multi-Infarct, Dementia—Semantic, Dementia—Subcortical, Dementia With Lewy Bodies, Demyelination diseases, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Distal hereditary motor neuronopathies, Dravet Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalomyelitis, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Episodic ataxia, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Faber's disease, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Friedreich's Ataxia, Frontotemporal Dementia, Gaucher Disease, Generalized Gangliosidoses (GM1, GM2), Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-Barré Syndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease, Hurler syndrome, Hydranencephaly, Hydrocephalus, Hydrocephalus—Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kluver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lichtheim's disease, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, Lyme Disease—Neurological Complications, Lysosomal storage disorders, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Mitochondrial DNA depletion syndromes, Moebius Syndrome, Monomelic Amyotrophy, Morvan Syndrome, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myelitis, Myoclonic Encephalopathy of Infants, Myoclonus, Myoclonus epilepsy, Myopathy, Myopathy—Congenital, Myopathy—Thyrotoxic, Myotonia, Myotonia Congenita, Narcolepsy, NARP (neuropathy, ataxia and retinitis pigmentosa), Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurodegenerative disease, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, Neurological Consequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathic pain, Neuropathy—Hereditary, Neuropathy, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain—Chronic, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Peroneal muscular atrophy, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive bulbar palsy, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Muscular Atrophy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudobulbar palsy, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease—Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjögren's Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Ataxia, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Sporadic ataxia, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central and Peripheral Nervous Systems, Vitamin B12 deficiency, Von Economo's Disease, Von Hippel-Lindau Disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman's Disease, X-Linked Spinal and Bulbar Muscular Atrophy.

Methods of Treatment of Neurological Disease TRACER AAV Particles Encoding Protein Payloads

Provided in the present disclosure are methods for introducing the TRACER AAV particles of the present disclosure into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for an increase in the production of target mRNA and protein to occur. In some aspects, the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.

Disclosed in the present disclosure are methods for treating neurological disease associated with insufficient function/presence of a target protein (e.g., ApoE, FXN) in a subject in need of treatment. The method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising TRACER AAV particles of the present disclosure. As a non-limiting example, the TRACER AAV particles can increase target gene expression, increase target protein production, and thus reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via systemic administration. In one embodiment, the systemic administration is intravenous injection.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject. In other embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to a CNS tissue of a subject (e.g., putamen, thalamus or cortex of the subject).

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via intraparenchymal injection. Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.

In one embodiment, the composition comprising the TRACER AAV particles of the present disclosure is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.

In one embodiment, the TRACER AAV particles of the present disclosure may be delivered into specific types of targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.

In one embodiment, the TRACER AAV particles of the present disclosure may be delivered to neurons in the putamen, thalamus and/or cortex.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for neurological disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for tauopathies.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Alzheimer's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Amyotrophic Lateral Sclerosis.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Huntington's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Parkinson's Disease.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for Friedreich's Ataxia.

In some embodiments, the TRACER AAV particles of the present disclosure may be used as a therapy for chronic or neuropathic pain.

In one embodiment, administration of the TRACER AAV particles described herein to a subject may increase target protein levels in a subject. The target protein levels may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the protein levels of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the proteins levels of a target protein by at least 40%. As a non-limiting example, a subject may have an increase of 10% of target protein. As a non-limiting example, the TRACER AAV particles may increase the protein levels of a target protein by fold increases over baseline. In one embodiment, TRACER AAV particles lead to 5-6 times higher levels of a target protein.

In one embodiment, administration of the TRACER AAV particles described herein to a subject may increase the expression of a target protein in a subject. The expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein by at least 40%.

In one embodiment, intravenous administration of the TRACER AAV particles described herein to a subject may increase the CNS expression of a target protein in a subject. The expression of the target protein may be increased by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein in the CNS by at least 50%. As a non-limiting example, the TRACER AAV particles may increase the expression of a target protein in the CNS by at least 40%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein expression in astrocytes in order to treat a neurological disease. Target protein in astrocytes may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in microglia. The increase of target protein in microglia may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in cortical neurons. The increase of target protein in the cortical neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in hippocampal neurons. The increase of target protein in the hippocampal neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles may be used to increase target protein in DRG and/or sympathetic neurons. The increase of target protein in the DRG and/or sympathetic neurons may be, independently, increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein in sensory neurons in order to treat neurological disease. Target protein in sensory neurons may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In some embodiments, the TRACER AAV particles of the present disclosure may be used to increase target protein and reduce symptoms of neurological disease in a subject. The increase of target protein and/or the reduction of symptoms of neurological disease may be, independently, altered (increased for the production of target protein and reduced for the symptoms of neurological disease) by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles of the present disclosure may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.

In one embodiment, the TRACER AAV particles of the present disclosure may be used to improve performance on any assessment used to measure symptoms of neurological disease. Such assessments include, but are not limited to ADAS-cog (Alzheimer Disease Assessment Scale—cognitive), MNISE (Mini-Mental State Examination), GDS (Geriatric Depression Scale), FAQ (Functional Activities Questionnaire), ADL (Activities of Daily Living), GPCOG (General Practitioner Assessment of Cognition), Mini-Cog, AMTS (Abbreviated Mental Test Score), Clock-drawing test, 6-CIT (6-item Cognitive Impairment Test), TYM (Test Your Memory), MoCa (Montreal Cognitive Assessment), ACE-R (Addenbrookes Cognitive Assessment), MIS (Memory Impairment Screen), BADLS (Bristol Activities of Daily Living Scale), Barthel Index, Functional Independence Measure, Instrumental Activities of Daily Living, IQCODE (Informant Questionnaire on Cognitive Decline in the Elderly), Neuropsychiatric Inventory, The Cohen-Mansfield Agitation Inventory, BEHAVE-AD, EuroQol, Short Form-36 and/or MBR Caregiver Strain Instrument, or any of the other tests as described in Sheehan B (Ther Adv Neurol Disord. 5(6):349-358 (2012)), the contents of which are herein incorporated by reference in their entirety.

In some embodiments, the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.

The TRACER AAV particles encoding the target protein may be used in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

Therapeutic agents that may be used in combination with the TRACER AAV particles of the present disclosure can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation. As a non-limiting example, the combination therapy may be in combination with one or more neuroprotective agents such as small molecule compounds, growth factors and hormones which have been tested for their neuroprotective effect on motor neuron degeneration.

Compounds tested for treating neurological disease which may be used in combination with the TRACER AAV particles described herein include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3 (3 (lithium) or PP2A, immunization with Aβ peptides or tau phospho-epitopes, anti-tau or anti-amyloid antibodies, dopamine-depleting agents (e.g., tetrabenazine for chorea), benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity, and/or spasticity), amino acid precursors of dopamine (e.g., levodopa for rigidity), skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and/or spasticity), inhibitors for acetylcholine release at the neuromuscular junction to cause muscle paralysis (e.g., botulinum toxin for bruxism and/or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and/or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and/or irritability, clozapine for treatment-resistant psychosis, aripiprazole for psychosis with prominent negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtazapine, venlafaxine for depression, anxiety, obsessive compulsive behavior and/or irritability), hypnotics (e.g., xopiclone and/or zolpidem for altered sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania) and mood stabilizers (e.g., lithium for mania or hypomania).

Neurotrophic factors may be used in combination therapy with the TRACER AAV particles of the present disclosure for treating neurological disease. Generally, a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron. In some embodiments, the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment. Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.

In one aspect, the TRACER AAV particle described herein may be co-administered with TRACER AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the contents of which are incorporated herein by reference in their entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, administration of the TRACER AAV particles to a subject will increase the expression of a target protein in a subject and the increase of the expression of the target protein will reduce the effects and/or symptoms of neurological disease in a subject.

As a non-limiting example, the target protein may be an antibody, or fragment thereof.

TRACER AAV Particles Comprising RNAi Agents or Modulatory Polynucleotides

Provided in the present disclosure are methods for introducing the TRACER AAV particles of the disclosure, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules into cells, the method comprising introducing into said cells any of the vectors in an amount sufficient for degradation of a target mRNA to occur, thereby activating target-specific RNAi in the cells. In some aspects, the cells may be neurons such as but not limited to, motor, hippocampal, entorhinal, thalamic, cortical, sensory, sympathetic, or parasympathetic neurons, and glial cells such as astrocytes, microglia, and/or oligodendrocytes.

Disclosed in the present disclosure are methods for treating neurological diseases associated with dysfunction of a target protein in a subject in need of treatment. The method optionally comprises administering to the subject a therapeutically effective amount of a composition comprising TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules. As a non-limiting example, the siRNA molecules can silence target gene expression, inhibit target protein production, and reduce one or more symptoms of neurological disease in the subject such that the subject is therapeutically treated.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure comprising a viral genome encoding one or more siRNA molecules comprise an AAV capsid that allows for enhanced transduction of CNS and/or PNS cells after intravenous administration.

In some embodiments, the composition comprising the TRACER AAV particles of the present disclosure with a viral genome encoding at least one siRNA molecule is administered to the central nervous system of the subject. In other embodiments, the composition comprising the TRACER AAV particles of the present disclosure is administered to a tissue of a subject (e.g., putamen, thalamus or cortex of the subject).

In one embodiment, the composition comprising the TRACER AAV particles of the disclosure, comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via systemic administration. In one embodiment, the systemic administration is intravenous injection.

In one embodiment, the composition comprising the TRACER AAV particles of the disclosure comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection. Non-limiting examples of intraparenchymal injections include intraputamenal, intracortical, intrathalamic, intrastriatal, intrahippocampal or into the entorhinal cortex.

In one embodiment, the composition comprising the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules is administered to the central nervous system of the subject via intraparenchymal injection and intravenous injection.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered into specific types or targeted cells, including, but not limited to, thalamic, hippocampal, entorhinal, cortical, motor, sensory, excitatory, inhibitory, sympathetic, or parasympathetic neurons; glial cells including oligodendrocytes, astrocytes and microglia; and/or other cells surrounding neurons such as T cells.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be delivered to neurons in the putamen, thalamus, and/or cortex.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for neurological disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for tauopathies.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Alzheimer' s Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Amyotrophic Lateral Sclerosis.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Huntington's Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Parkinson's Disease.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used as a therapy for Friedreich's Ataxia.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower target protein levels in a subject. The target protein levels may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the protein levels of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the proteins levels of a target protein by at least 40%.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in a subject. The expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 40%.

In one embodiment, the administration of TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules to a subject may lower the expression of a target protein in the CNS of a subject. The expression of a target protein may be lowered by about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 40-90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 60-80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 80-100%, 90-95%, 90-100% or 95-100% in a subject such as, but not limited to, the CNS, a region of the CNS, or a specific cell of the CNS of a subject. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 50%. As a non-limiting example, the TRACER AAV particles may lower the expression of a target protein by at least 40%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in astrocytes in order to treat neurological disease. Target protein in astrocytes may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. Target protein in astrocytes may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in microglia. The suppression of the target protein in microglia may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress target protein in cortical neurons. The suppression of a target protein in cortical neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in hippocampal neurons. The suppression of a target protein in the hippocampal neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in DRG and/or sympathetic neurons. The suppression of a target protein in the DRG and/or sympathetic neurons may be, independently, suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. The reduction may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein in sensory neurons in order to treat neurological disease. Target protein in sensory neurons may be suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%. Target protein in the sensory neurons may be reduced may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to suppress a target protein and reduce symptoms of neurological disease in a subject. The suppression of target protein and/or the reduction of symptoms of neurological disease may be, independently, reduced or suppressed by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 5-55%, 5-60%, 5-65%, 5-70%, 5-75%, 5-80%, 5-85%, 5-90%, 5-95%, 10-20%, 10-25%, 10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 10-55%, 10-60%, 10-65%, 10-70%, 10-75%, 10-80%, 10-85%, 10-90%, 10-95%, 15-25%, 15-30%, 15-35%, 15-40%, 15-45%, 15-50%, 15-55%, 15-60%, 15-65%, 15-70%, 15-75%, 15-80%, 15-85%, 15-90%, 15-95%, 20-30%, 20-35%, 20-40%, 20-45%, 20-50%, 20-55%, 20-60%, 20-65%, 20-70%, 20-75%, 20-80%, 20-85%, 20-90%, 20-95%, 25-35%, 25-40%, 25-45%, 25-50%, 25-55%, 25-60%, 25-65%, 25-70%, 25-75%, 25-80%, 25-85%, 25-90%, 25-95%, 30-40%, 30-45%, 30-50%, 30-55%, 30-60%, 30-65%, 30-70%, 30-75%, 30-80%, 30-85%, 30-90%, 30-95%, 35-45%, 35-50%, 35-55%, 35-60%, 35-65%, 35-70%, 35-75%, 35-80%, 35-85%, 35-90%, 35-95%, 40-50%, 40-55%, 40-60%, 40-65%, 40-70%, 40-75%, 40-80%, 40-85%, 40-90%, 40-95%, 45-55%, 45-60%, 45-65%, 45-70%, 45-75%, 45-80%, 45-85%, 45-90%, 45-95%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 70-80%, 70-85%, 70-90%, 70-95%, 75-85%, 75-90%, 75-95%, 80-90%, 80-95%, or 90-95%.

In one embodiment, the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used to reduce the decline of functional capacity and activities of daily living as measured by a standard evaluation system such as, but not limited to, the total functional capacity (TFC) scale.

In some embodiments, the present composition is administered as a solo therapeutic or as combination therapeutic for the treatment of neurological disease.

The TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules may be used in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

Therapeutic agents that may be used in combination with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules can be small molecule compounds which are antioxidants, anti-inflammatory agents, anti-apoptosis agents, calcium regulators, antiglutamatergic agents, structural protein inhibitors, compounds involved in muscle function, and compounds involved in metal ion regulation.

Compounds tested for treating neurological disease which may be used in combination with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules include, but are not limited to, cholinesterase inhibitors (donepezil, rivastigmine, galantamine), NMDA receptor antagonists such as memantine, anti-psychotics, anti-depressants, anti-convulsants (e.g., sodium valproate and levetiracetam for myoclonus), secretase inhibitors, amyloid aggregation inhibitors, copper or zinc modulators, BACE inhibitors, inhibitors of tau aggregation, such as Methylene blue, phenothiazines, anthraquinones, n-phenylamines or rhodamines, microtubule stabilizers such as NAP, taxol or paclitaxel, kinase or phosphatase inhibitors such as those targeting GSK3β (lithium) or PP2A, immunization with Aβ peptides or tau phospho-epitopes, anti-tau or anti-amyloid antibodies, dopamine-depleting agents (e.g., tetrabenazine for chorea), benzodiazepines (e.g., clonazepam for myoclonus, chorea, dystonia, rigidity, and/or spasticity), amino acid precursors of dopamine (e.g., levodopa for rigidity), skeletal muscle relaxants (e.g., baclofen, tizanidine for rigidity and/or spasticity), inhibitors for acetylcholine release at the neuromuscular junction to cause muscle paralysis (e.g., botulinum toxin for bruxism and/or dystonia), atypical neuroleptics (e.g., olanzapine and quetiapine for psychosis and/or irritability, risperidone, sulpiride and haloperidol for psychosis, chorea and/or irritability, clozapine for treatment-resistant psychosis, aripiprazole for psychosis with prominent negative symptoms), selective serotonin reuptake inhibitors (SSRIs) (e.g., citalopram, fluoxetine, paroxetine, sertraline, mirtazapine, venlafaxine for depression, anxiety, obsessive compulsive behavior and/or irritability), hypnotics (e.g., xopiclone and/or zolpidem for altered sleep-wake cycle), anticonvulsants (e.g., sodium valproate and carbamazepine for mania or hypomania) and mood stabilizers (e.g., lithium for mania or hypomania).

Neurotrophic factors may be used in combination therapy with the TRACER AAV particles comprising a viral genome with a nucleic acid sequence encoding one or more siRNA molecules for treating neurological disease. Generally, a neurotrophic factor is defined as a substance that promotes survival, growth, differentiation, proliferation and/or maturation of a neuron, or stimulates increased activity of a neuron. In some embodiments, the present methods further comprise delivery of one or more trophic factors into the subject in need of treatment. Trophic factors may include, but are not limited to, IGF-I, GDNF, BDNF, CTNF, VEGF, Colivelin, Xaliproden, Thyrotrophin-releasing hormone and ADNF, and variants thereof.

In one aspect, the TRACER AAV particle encoding the nucleic acid sequence for the at least one siRNA duplex targeting the gene of interest may be co-administered with TRACER AAV particles expressing neurotrophic factors such as AAV-IGF-I (See e.g., Vincent et al., Neuromolecular medicine, 2004, 6, 79-85; the content of which is incorporated herein by reference in its entirety) and AAV-GDNF (See e.g., Wang et al., J Neurosci., 2002, 22, 6920-6928; the contents of which are incorporated herein by reference in their entirety).

In one embodiment, administration of the TRACER AAV particles to a subject will reduce the expression of a target protein in a subject and the reduction of expression of the target protein will reduce the effects and/or symptoms of neurological disease in a subject.

DEFINITIONS

Adeno-associated virus: As used herein, the term “adeno-associated virus” or “AAV” refers to members of the Dependovirus genus comprising any particle, sequence, gene, protein, or component derived therefrom.

AAV Particle: As used herein, an “AAV particle” is a virus which comprises a capsid and a viral genome with at least one payload region and at least one ITR. As used herein “AAV particles of the disclosure” are AAV particles comprising a parent capsid sequence with at least one targeting peptide insert. AAV particles of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In addition, the AAV particle may be replication defective and/or targeted. In one embodiment, the AAV particle may have a targeting peptide inserted into the capsid to enhance tropism for a desired target tissue. It is to be understood that reference to the AAV particles of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.

Administering: As used herein, the term “administering” refers to providing a pharmaceutical agent or composition to a subject.

Amelioration: As used herein, the term “amelioration” or “ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of neurodegeneration disorder, amelioration includes the reduction of neuron loss.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically engineered animal, or a clone.

Antisense strand: As used herein, the term “the antisense strand” or “the first strand” or “the guide strand” of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of a gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process.

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Capsid: As used herein, the term “capsid” refers to the protein shell of a virus particle.

Complementary and substantially complementary: As used herein, the term “complementary” refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be complementary to adenine. However, when a U is denoted in the context of the present disclosure, the ability to substitute a T is implied, unless otherwise stated. Perfect complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bond with each other. For example, for two 20-mers, if only two base pairs on each strand can form a hydrogen bond with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity. As used herein, the term “substantially complementary” means that the siRNA has a sequence (e.g., in the antisense strand) which is sufficient to bind the desired target mRNA, and to trigger the RNA silencing of the target mRNA.

Control Elements: As used herein, “control elements”, “regulatory control elements” or “regulatory sequences” refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and/or translated in an appropriate host cell.

Delivery: As used herein, “delivery” refers to the act or manner of delivering an AAV particle, a compound, substance, entity, moiety, cargo or payload.

Element: As used herein, the term “element” refers to a distinct portion of an entity. In some embodiments, an element may be a polynucleotide sequence with a specific purpose, incorporated into a longer polynucleotide sequence.

Encapsulate: As used herein, the term “encapsulate” means to enclose, surround or encase. As an example, a capsid protein often encapsulates a viral genome.

Engineered: As used herein, embodiments of the disclosure are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

Effective Amount: As used herein, the term “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.

Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

Feature: As used herein, a “feature” refers to a characteristic, a property, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least one AAV particle (active ingredient) and an excipient, and/or an inactive ingredient.

Fragment: A “fragment,” as used herein, refers to a portion. For example, an antibody fragment may comprise a CDR, or a heavy chain variable region, or a scFv, etc.

Functional: As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

Gene expression: The term “gene expression” refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide. For clarity, when reference is made to measurement of “gene expression”, this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.

Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). In accordance with the disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. In accordance with the disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; the contents of each of which are incorporated herein by reference in their entirety. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibit expression of a gene” means to cause a reduction in the amount of an expression product of the gene. The expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression may be determined using standard techniques for measuring mRNA or protein.

Insert: As used herein the term “insert” may refer to the addition of a targeting peptide sequence to a parent AAV capsid sequence. An “insertion” may result in the replacement of one or more amino acids of the parent AAV capsid sequence. Alternatively, an insertion may result in no changes to the parent AAV capsid sequence beyond the addition of the targeting peptide sequence.

Inverted terminal repeat: As used herein, the term “inverted terminal repeat” or “ITR” refers to a cis-regulatory element for the packaging of polynucleotide sequences into viral capsids.

Library: As used herein, the term “library” refers to a diverse collection of linear polypeptides, polynucleotides, viral particles, or viral vectors. As examples, a library may be a DNA library or an AAV capsid library.

Neurological disease: As used herein, a “neurological disease” is any disease associated with the central or peripheral nervous system and components thereof (e.g., neurons).

Naturally Occurring: As used herein, “naturally occurring” or “wild-type” means existing in nature without artificial aid, or involvement of the hand of man.

Open reading frame: As used herein, “open reading frame” or “ORF” refers to a sequence which does not contain a stop codon in a given reading frame.

Parent sequence: As used herein, a “parent sequence” is a nucleic acid or amino acid sequence from which a variant is derived. In one embodiment, a parent sequence is a sequence into which a heterologous sequence is inserted. In other words, a parent sequence may be considered an acceptor or recipient sequence. In one embodiment, a parent sequence is an AAV capsid sequence into which a targeting sequence is inserted.

Particle: As used herein, a “particle” is a virus comprised of at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.

Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

Payload region: As used herein, a “payload region” is any nucleic acid sequence (e.g., within the viral genome) which encodes one or more “payloads” of the disclosure. As non-limiting examples, a payload region may be a nucleic acid sequence within the viral genome of an AAV particle, which encodes a payload, wherein the payload is an RNAi agent or a polypeptide. Payloads of the present disclosure may be, but are not limited to, peptides, polypeptides, proteins, antibodies, RNAi agents, etc.

Peptide: As used herein, “peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Preventing: As used herein, the term “preventing” or “prevention” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.

Region: As used herein, the term “region” refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and/or a cluster of epitopes. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to proteins, terminal regions may comprise N- and/or C-termini.

In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term “terminal region” refers to regions located at the ends or termini of a given agent. When referring to polynucleotides, terminal regions may comprise 5′ and/or 3′ termini.

RNA or RNA molecule: As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides; the term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). The term “mRNA” or “messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.

RNA interfering or RNAi: As used herein, the term “RNA interfering” or “RNAi” refers to a sequence specific regulatory mechanism mediated by RNA molecules which results in the inhibition or interfering or “silencing” of the expression of a corresponding protein-coding gene. RNAi has been observed in many types of organisms, including plants, animals and fungi. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. RNAi is controlled by the RNA-induced silencing complex (RISC) and is initiated by short/small dsRNA molecules in cell cytoplasm, where they interact with the catalytic RISC component argonaute. The dsRNA molecules can be introduced into cells exogenously. Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves dsRNAs to produce double-stranded fragments of 21-25 base pairs with a few unpaired overhang bases on each end. These short double stranded fragments are called small interfering RNAs (siRNAs).

RNAi agent: As used herein, the term “RNAi agent” refers to an RNA molecule, or its derivative, that can induce inhibition, interfering, or “silencing” of the expression of a target gene and/or its protein product. An RNAi agent may knock-out (virtually eliminate or eliminate) expression, or knock-down (lessen or decrease) expression. The RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA.

Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

Self-complementary viral particle: As used herein, a “self-complementary viral particle” is a particle comprised of at least two components, a protein capsid and a self-complementary viral genome enclosed within the capsid.

Sense Strand: As used herein, the term “the sense strand” or “the second strand” or “the passenger strand” of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand. The antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure. As used herein, a “siRNA duplex” includes a siRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a siRNA strand having sufficient complementarity to form a duplex with the other siRNA strand.

Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

Short interfering RNA or siRNA: As used herein, the terms “short interfering RNA,” “small interfering RNA” or “siRNA” refer to an RNA molecule (or RNA analog) comprising between about 5-60 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi. Preferably, a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogs, such as between about 16-25 nucleotides (or nucleotide analogs), between about 18-23 nucleotides (or nucleotide analogs), between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs), between about 19-25 nucleotides (or nucleotide analogs), and between about 19-24 nucleotides (or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising 5-23 nucleotides, preferably 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, or as few as 5 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, e.g., 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi or translational repression absent further processing, e.g., enzymatic processing, to a short siRNA. siRNAs can be single stranded RNA molecules (ss-siRNAs) or double stranded RNA molecules (ds-siRNAs) comprising a sense strand and an antisense strand which hybridized to form a duplex structure called an siRNA duplex.

Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Targeting peptide: As used herein, a “targeting peptide” refers to a peptide of 3-20 amino acids in length. These targeting peptides may be inserted into, or attached to, a parent amino acid sequence to alter the characteristics (e.g., tropism) of the parent protein. As a non-limiting example, the targeting peptide can be inserted into an AAV capsid sequence for enhanced targeting to a desired cell-type, tissue, organ or organism. It is to be understood that a targeting peptide is encoded by a targeting polynucleotide which may similarly be inserted into a parent polynucleotide sequence. Therefore, a “targeting sequence” refers to a peptide or polynucleotide sequence for insertion into an appropriate parent sequence (amino acid or polynucleotide, respectively).

Target Cells: As used herein, “target cells” or “target tissue” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose.

Therapeutically effective outcome: As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example, “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

Vector: As used herein, the term “vector” refers to any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. In some embodiments, vectors may be plasmids. In some embodiments, vectors may be viruses. An AAV particle is an example of a vector. Vectors of the present disclosure may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parent or reference sequences. The heterologous molecule may be a polynucleotide and/or a polypeptide.

Viral Genome: As used herein, the terms “viral genome” or “vector genome” refer to the nucleic acid sequence(s) encapsulated in an AAV particle. A viral genome comprises a nucleic acid sequence with at least one payload region encoding a payload and at least one ITR.

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

The present disclosure is further illustrated by the following non-limiting examples.

EXAMPLES Example 1. TRACER Proof of Concept: Promoter Selection

Proof-of-concept experiments were conducted by placing the genes encoding an AAV9 peptide display capsid library under the control of either the neuron-specific synapsin promoter (SYN) or the astrocyte-specific GFAP promoter. Following intravenous administration to C57BL/6 mice, RNA was recovered from brain tissue and used for further library evolution. Next-generation sequencing (NGS) showed sequence convergence between animals after only two rounds of selection. Interestingly, several variants highly similar to the PHP.eB capsid were recovered, suggesting that our method allowed a rapid selection of high-performance capsids. A subset of capsids having peptide sequences with high CNS enrichment was selected for further study. It is understood that any promoter may be selected depending on the desired tropism. Examples of such promoters are found in Table 3.

TABLE 3 Promoters, tissue and cell type Promoter name Tissue Cell type B29 promoter Blood B cells Immunoglobulin heavy chain Blood B cells promoter CD45 promoter Blood Hematopoietic Mouse INF-β promoter Blood Hematopoietic CD45 SV40/CD45 promoter Blood Hematopoietic WASP promoter Blood Hematopoietic CD43 promoter Blood Leuko & Platelets CD43 SV40/CD43 promoter Blood Leuko & Platelets CD68 promoter Blood Macrophages GPIIb promoter Blood Megakaryocyte CD14 promoter Blood Monocytes CD2 promoter Blood T cells Osteocalcin Bone Osteoblasts Bone sialoprotein Bone Osteoblasts OG-2 promoter Bone Osteoblasts, odontoblasts GFAP promoter Brain Astrocytes Vga Brain GABAergic neurons Vglut2 Brain glutamatergic neurons NSE/RU5′ promoter Brain Neurons SYN1 promoter Brain Neurons Neurofilament light chain Brain Neurons VGF Brain Neurons Nestin Brain NSC Chx10 Eye All retinal neurons PrP Eye All retinal neurons Dkk3 Eye All retinal neurons Math5 Eye Amacrine and horizontal cells Ptf1a Eye Amacrine and horizontal cells Pcp2 Eye Bipolar cells Nefh Eye Ganglion cells gamma-synuclein gene Eye ganglion cells (SNCG) Grik4 Eye GC Pdgfra Eye GC and ONL Müller cells Chat Eye GC/Amacrine cells Thy 1.2 Eye GC/neural retina hVmd2 Eye INL Müller cells Thy 1 Eye INL Müller cells Modified αA-crystallin Eye Lens/neural retina hRgp Eye M- and S-cone mMo Eye M-cone Opn4 Eye Melanopsin-expressing GC RLBP1 Eye Muller cells Glast Eye Müller cells Foxg1 Eye Müller cells hVmd2 Eye Müller cells/optic nerve/ INL Trp1 Eye Neural retina Six3 Eye Neural retina cx36 Eye Neurons Grm6 - SV40 eukaryotic Eye ON bipolar promoter hVmd2 Eye Optic nerve Dct Eye Pigmented cells Rpc65 Eye Retinal pigment epithelium mRho Eye Rod Irbp Eye Rod hRho Eye Rod Pcp2 Eye Rod bipolar cells Rhodopsin Eye Rod Photoreceptors mSo Eye S-cone MLC2v promoter Heart Cardiomyocyte αMHC promoter Heart Cardiomyocyte rat troponin T (Tnnt2) Heart Cardiomyocyte Tie2 Heart Endothelial Tcf21 Heart Fibroblasts ECAD Kidney Collecting duct NKCC2 Kidney Loop of Henle KSPC Kidney Nephron NPHS1 Kidney Podocyte SGLT2 Kidney Proximal tubular cells SV40/bAlb promoter Liver hepatocytes SV40/hAlb promoter Liver hepatocytes Hepatitis B virus core Liver hepatocytes promoter Alpha fetoprotein Liver hepatocytes Surfactant protein B promoter Lung AT II cells and Clara cells Surfactant protein C promoter Lung AT II cells and Clara cells Desmin Muscle Muscle stem cells + Myocytes Mb promoter Muscle Myocyte Myosin Muscle Myocyte Dystrophin Muscle Myocyte dMCK and tMCK Muscle Myocytes Elastase-1 promoter Pancreas Acinar cells PDX1 promoter Pancreas Beta cells Insulin promoter Pancreas langherans Slco1c1 Vasculature BBB Endothelial tie Vasculature Endothelial cadherin Vasculature Endothelial ICAM-2 Vasculature Endothelial claudin 1 Vasculature Endothelial Cldn5 Vasculature Endothelial Flt-1 promoter Vasculature Endothelial Endoglin promoter Vasculature Endothelial

Capsid pools were injected to three rodent species, followed by RNA enrichment analysis for characterization of transduction efficiency in neurons or astrocytes and cross-species performance. Top-ranking capsids were then individually tested and several variants showed CNS transduction similar to or higher than the PHP.eB benchmark. These results suggest that the TRACER platform allows rapid in vivo evolution of AAV capsids in non-transgenic animals with a high degree of tropism improvement. The following examples illustrate the findings in more detail.

Example 2. Generation of an AAV Vectors Capable of Capsid mRNA Expression in the Absence of Helper Virus

In order to perform cell type- and transduction-restricted in vivo evolution of AAV capsid libraries, a capsid library system was engineered in which the capsid mutant gene can be transcribed in the absence of a helper virus, in a specific cell type. In the wild-type AAV virus, the mRNA encoding the capsid proteins VP1, VP2 and VP3, as well as the AAP accessory protein, are expressed by the P40 promoter located in the 3′ region of the REP gene (FIG. 1A), that is only active in the presence of the REP protein as well as the helper virus functions (Berns et al., 1996). In order to allow expression of the capsid mRNA in animal tissue or in cultured cells, another promoter must be inserted upstream or downstream of the CAP gene. Because of the limited packaging capacity of the AAV capsid, a portion of the REP gene must be deleted to accommodate the extra promoter insertion, and the REP gene has to be provided in trans by another plasmid to allow virus production. The minimal viral sequence required for high titer AAV production was determined by introducing a CMV promoter at various locations upstream of the CAP gene of AAV9 (FIG. 1B). The REP protein was provided in trans by the pREP2 plasmid obtained by deleting the CAP gene from a REP2-CAP2 packaging vector using EcoNI and ClaI (SEQ. ID NO:4). For small-scale virus production test, HEK-293T cells grown in DMEM supplemented with 5% FBS and 1× pen/strep were plated in 15-cm dishes and co-transfected with 15 ug of pHelper (pFdelta6) plasmid, 10 ug pREP2 plasmid and lug ITR-CMV-CAP plasmid using calcium phosphate transfection. After 72 hours, cells were harvested by scraping, pelleted by a brief centrifugation and suspended in 1 ml of a buffer containing 10 mM Tris and 2 mM MgCl2. Cells were lysed by addition of triton X-100 to 0.1% final concentration and treated with 50U of benzonase for 1 hour. Virus from the supernatants was precipitated with 8% polyethylene glycol and 0.5M NaCl, suspended in 1 ml of 10 mM TRIS-2mM MgCl2 and combined with the cell lysate. The pooled virus was adjusted to 0.5M NaCl, cleared by centrifugation for 15 minutes at 4,000×g and fractionated on a step iodixanol gradient of 15%, 25%, 40% and 60% for 3 hours at 40,000prm (Zolotukhin et al., 1999). The 40% fraction containing the purified AAV particles was harvested and viral titers were measured by real-time PCR using a Taqman primer/probe mix specific for the 3′-end of REP, shared by all the constructs. Virus yields were significantly lower than the fully wild-type ITR-REP2-CAP9-ITR used as a reference (1.7% to 8.8%), but the CMV-BstEII construct allowed the highest yields of all three CMV constructs. See FIG. 2. The CMV-HindIII construct, in which most of the P40 promoter sequence is deleted, generated the lowest yield (1.7% of wtAAV9), indicating that even the potent CMV promoter cannot replace the P40 promoter without a severe drop in virus yields. Following these observations, the BstEII architecture (SEQ. ID NO:5), which preserves the minimal P40 sequence and the CAP mRNA splice donor, was used in all further experiments.

The REP-expressing plasmid was then improved by preserving the AAP reading frame together with a large portion of the capsid gene from the REP2-CAP9 helper vector, which may contain sequences necessary for the regulation of CAP transcription and/or splicing. In order to eliminate the capsid coding potential of the vector, a C-terminus fragment of the capsid gene was deleted by a triple cut with the MscI restriction enzyme followed by self-ligation, in order to obtain the pREP-AAP plasmid (FIG. 3A, SEQ. ID NO:6).

An iteration of this construct was engineered by introducing premature stop codons immediately after the start codons of VP1, VP2 and VP3, without perturbing the amino acid sequence of the colinear AAP reading frame (FIG. 3A). This construct was named pREP-3stop (SEQ. ID NO:7). A neuron-specific syn-CAPS vector (SEQ. ID NO:8) was derived from the CMV9-BstEII plasmid by swapping the CMV promoter with the neuron-specific human synapsin 1 promoter.

Production efficiency of this Syn-CAPS was tested as described previously using pREP, pREP-AAP or pREP-3stop plasmid to supply REP in trans. As shown in FIG. 3B, the REP plasmids harboring a longer capsid sequence as well as AAP increased virus yields by approximately 3-fold compared to the pREP plasmid. Virus titers obtained with the pREP-AAP or pREP-3stop vectors reached ˜30% of wild-type AAV9. An important concern with plasmids harboring long homologous regions is the potential for unwanted recombination with the ITR-CAP vector, that would reconstitute a wild-type ITR-REP-CAP vector and contaminate combinatorial libraries.

To evaluate the risk of wild-type virus reconstitution, the viral preparations obtained in FIG. 3B were subjected to real-time PCR with a Taqman probe located in the N terminus of REP. The percentage of capsids containing a detectable full-length REP was less than 0.03% of wild-type virus (FIG. 3C), which was even lower than the routinely detected 0.1% illegitimate REP-CAP packaging occurring in most recombinant AAV preparations obtained from 293T cell transfection (FIG. 3C, our unpublished observations). Because the premature stop codons of the pREP-3 stop vector offer an extra layer of safety against potential reconstitution of wild-type capsids and prevents the translation of truncated capsid proteins, the 3stop plasmid was used for all subsequent studies.

Following this, the feasibility of RNA-driven biopanning in C57BL/6 mice using AAV9-packaged vectors where the AAV9 capsid gene is driven by the CMV promoter, the Synapsin promoter or the astrocyte-specific GFabc1D promoter (SEQ. ID NO:9), thereafter referred to as GFAP promoter (Brenner et al., 2008) was tested (FIG. 4A). The three vectors were produced in HEK-293T cells as previously described and analyzed by PAGE-silver stain. As shown in FIG. 4B, all vectors showed a normal ratio of VP1, VP2 and VP3 capsid proteins, indicating that the particular promoter architecture does not disrupt the balance of capsid protein expression. Six-week old male C57BL/6 mice were injected intravenously with 1e12 VG per mouse and sacrificed after 28 days. DNA biodistribution and capsid mRNA expression were tested in the brain, liver and heart tissues.

Total DNA was extracted from brain, liver and heart tissues using Qiagen DNeasy Blood and Tissue columns, and viral DNA was quantified by real-time PCR using a Taqman probe located in the VP3 N-terminal region. DNA abundance was normalized using a pre-designed probe detecting the single-copy transferrin receptor gene (Life Technologies ref. 4458366). Viral DNA was highly abundant in the liver and to a lower extent in the heart. The DNA distribution did not show any noticeable difference between the three vectors (FIG. 4C). RNA was extracted with Qiagen RNeasy plus universal kit following manufacturer's instructions, then treated with ezDNAse (Qiagen) to remove residual DNA, and reverse transcribed with Superscript IV (Life technologies).

RNA expression was evaluated using the same VP3 probe used to quantify viral DNA and normalized using TBP as a reference RNA (Life technologies Mm01277042 m1). In the brain, the GFAP promoter allowed the strongest expression level, and the Synapsin promoter allowed a comparable expression as the potent CMV promoter. In the liver, all promoters resulted in a similar expression level, which could be the result of a leaky expression at very high copy number (FIG. 4D). In the heart, the cell type specificity of the Syn and GFAP promoters was evident, since they allowed only ˜3 and 10% of CMV expression, respectively despite of a similar DNA biodistribution.

Overall the experiment showed that mRNA from transduction-competent capsids could be recovered from various animal organs, including weakly transduced tissues such as the brain.

Example 3. AAV Vector Configuration

Various vector configurations were explored toward increasing RNA expression to maximize library recovery. The CMV promoter was replaced by a hybrid CMV enhancer/Chicken beta-actin promoter sequence (Niwa et al., 1991) and a potent cytomegalovirus-beta-globin hybrid intron derived from the AAV-MCS cloning vector (Stratagene) was inserted between the promoter sequence and the capsid gene, as introns have been shown to increase mRNA processing and stability (Powell et al., 2015). This resulted in the constructs CAG9 (SEQ. ID NO:10), SYNG9 (SEQ. ID NO:11) and GFAPG (SEQ. ID NO:12).

An inverted vector configuration was also tested where the helper-independent promoter was placed downstream of the capsid gene in reverse orientation, in order to avoid potential interference with the P40 promoter (FIG. 5A). This configuration allows the expression of an antisense capsid transcript in animal tissue. Because most polyadenylation signals (AATAAA) are orientation-dependent, it was hypothesized that the natural AAV capsid polyA would not prematurely terminate transcription when placed in reverse orientation. All constructs were co-transfected with pHelper and pREP-3 stop plasmids to generate AAV9-packaged virions that were used to transduce HEK-293T cells at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-transfection and reverse transcribed using the Quantitect kit (Qiagen).

PCR was performed with primers allowing amplification of the full-length capsid or a partial sequence localized close to the C-terminus (FIG. 5B). Overall, the presence of an intron had little influence on the expression from low-activity promoters Syn and GFAP, which indicates that mRNA splicing did not alleviate promoter repression in nonpermissive cells. The combination of the CMV enhancer with a Chicken beta-actin promoter and the hybrid intron allowed a significantly higher (>10-fold) mRNA expression compared to CMV promoter alone (FIGS. 5B, C).

When comparing endpoint PCR amplification between forward and inverted intronic vectors, a discrepancy was obvious between full-length and partial capsid amplicons (FIG. 5B, right-hand lanes), which led us to question the integrity of capsid RNA. When cDNA from inverted iCAG9 genome was amplified using primers flanking the full-length capsid, multiple low-molecular weight bands were detected, whereas the forward orientation vector allowed amplification of a single product with the expected length (FIG. 5D). Sanger sequencing of low-molecular weight amplicons showed that each band corresponded to an illegitimate splicing product from the antisense capsid RNA.

In light of these results, the forward tandem promoter architecture for subsequent experiments.

Splice-specific PCR amplification was tested to avoid amplification of residual DNA present in RNA preparations. Two candidate PCR primers overlapping the CMV/Globin exon-exon junction were designed and tested them for amplification of cDNA (spliced) or plasmid DNA (still containing the intron sequence). As shown in FIG. 5E, the GloSpliceF6 primer (SEQ. ID NO:13) allowed a fully specific amplification from cDNA without producing a detectable amplicon from the plasmid DNA sequence. This primer was used in subsequent assays to ascertain the absence of amplification from contaminating DNA.

Tandem constructs were then tested for potential interference of the P40 promoter with the cell-specific promoter placed upstream. For this, two series of AAV genomes were tested for transgene mRNA expression in HEK-293T cells. A series of transgenes where the GFP gene was placed immediately downstream of the CAG, SYNG or GFAPG promoter without P40 sequence were tested, and compared to the library constructs where AAV9 capsid was placed downstream of the P40 promoter (FIG. 6A). All genomes were packaged into the AAV9 capsid and used to infect HEK-293T at a MOI of 1e4 VG per cell. RNA was extracted 48 hours post-infection and transgene RNA was quantified by using a Taqman primer/probe mix specific for the spliced globin exon-exon junction. As shown in FIG. 6B, the expression from the CAG promoter was similar between the GFP and the P40-CAP9 constructs (2-fold lower in p40-CAP9, within the error margin of AAV titration). Expression from the synapsin promoter was drastically lower with both constructs and even lower for GFAP-driven mRNA (FIG. 6B). This was expected since HEK-293T cells are not permissive to Synapsin or GFAP promoter expression. Overall, this experiment confirmed that the presence of the P40 sequence did not alter the cell type specificity of synapsin or GFAP promoters.

This novel platform was termed TRACER (Tropism Redirection of AAV by Cell type-specific Expression of RNA). The TRACER platform solves the problems of standard methods including transduction and cell-type restrictions. (FIG. 7). Use of the TRACER system is well suited to capsid discovery where targeting peptide libraries are utilized. Screening of such a library may be conducted as outlined in FIG. 8.

While several variations of the AAV vectors which encode the capsids as payloads are taught herein, one canonical design is shown in FIG. 9B and in FIG. 12A and FIG. 12B.

Further advantages of the TRACER platform relate to the nature of the virus pool and the recovery of RNA only from fully transduced cells (FIG. 10). Consequently, capsid discovery can be accelerated in a manner that results in cell and/or tissue specific tropism (FIG. 11).

Example 4. Generation of Peptide Display Libraries and Cloning-Free Amplification

Several peptide display capsid libraries were generated by insertion of seven contiguous randomized amino acids into the surface-exposed hypervariable loop VIII region of AAV5, AAV6, or AAV-DJ8 capsids (FIG. 13 and FIG. 39) as well as AAV9 (FIG. 14). For AAV9 libraries, two extra libraries by modifying residues at positions −2, −1 and +1 of the insertion to match the flanking sequence of the highly neurotrophic PHP.eB vector (Chan et al., 2018). In order to facilitate the insertion of various loops and to prevent contamination by wild-type capsids, defective shuttle vectors were generated in which the C-terminal region of the capsid gene comprised between the loop VIII and the stop codon was deleted and replaced by a unique BsrGI restriction site (FIGS. 15A, B). Degenerate primers containing randomized NNK (K=T or G) sequences able to encode all amino acids were synthesized by IDT and used to amplify the missing capsid fragment using gBlock (IDT) double-stranded linear DNA as templates (SEQ. ID NO 14, 15, 16, 17). Linear PCR templates were preferred to plasmids in order to completely prevent the possibility of plasmid carryover in the PCR reaction. Amplicons containing the random library sequence (500 ng) were inserted in the shuttle plasmid linearized by BsrGI (2 ug) using 100 ul of NEBuilder HiFi DNA assembly master mix (NEB) during 30 minutes at 50° C. Unassembled linear templates were eliminated by addition of 5 ul of T5 exonuclease to the reaction and digestion for 30 minutes at 37° C. The entire reaction was purified with DNA Clean and Concentrator-5 and quantified with a nanodrop to estimate the efficiency of assembly. This method routinely allows the recovery of 0.5-1 ug assembled material.

gBlock templates were engineered by introducing silent mutations to remove unique restriction sites, to allow selective elimination of wild-type virus contaminants from the libraries by restriction enzyme treatment. As an example, AAV9 gBlock was engineered to remove BamHI and AfeI sites present in the parental sequence (SEQ. ID NO 17).

Example 5. Cloning Free Amplification

Transformation of assembled library DNA into competent bacteria represents a major bottleneck in library diversity, since even highly competent strains rarely exceed 1e7-1e8 colonies per transformation. By comparison, 100 nanograms of a 6-kilobase plasmid contain 1.5e10 DNA molecules. Therefore, bacterial transformation arbitrarily eliminates more than 99% of DNA species in a given pool. A cloning-free method was therefore created that allows >100-fold amplification of Gibson-assembled DNA while bypassing the bacterial transformation bottleneck (FIG. 16). A protocol based on rolling-circle amplification was optimized, which allows unbiased exponential amplification of circular DNA templates with an extremely low error rate (Hutchinson et al., 2005). One issue with rolling circle amplification is that it produces very large (˜70 kilobases on average) heavily branched concatemers that have to be cleaved into monomers for efficient cell transfection. This process can be accomplished by several methods, for example by using restriction enzymes to generate open-ended linear templates (Hutchinson et al., 2005, Huovinen, 2012), or CRE-Lox recombination to generates self-ligated circular templates (Huovinen et al., 2011). However, open-ended DNA is sensitive to degradation by cytoplasmic exonucleases, and the CRE recombination method showed relatively low efficiency (our unpublished observations). Therefore, an alternative monomer resolution method was chosen based on the use of TelN protelomerase (Rybchin et al., 1999), an enzyme that catalyzes the formation of closed-ended linear “dogbone” DNA monomers that are highly suitable for mammalian cell transfection (Heinrich et al., 2002).

To that end, the protelomerase recognition sequence TATCAGCACACAATTGCCCATTATACGC*GCGTATAATGGACTATTGTGTGCTGATA (SEQ ID NO: 176) was introduced outside both ITRs in all the BsrGI shuttle vectors used for capsid library insertion (the asterisk depicts the position were the two complementary strands get covalently linked to each other), in order to obtain the following plasmids: TelN-Syn9-BsrGI (SEQ ID NO 18), TelN-GFAP9-BsrGI (SEQ ID NO 19), TelN-Syn5-BsrGI (SEQ ID NO 20), TelN-GFAP5-BsrGI (SEQ ID NO 21), TelN-Syn6-BsrGI (SEQ ID NO 22), TelN-GFAP6-BsrGI (SEQ ID NO 23), TelN-SynDJ8-BsrGI (SEQ ID NO 24), TelN-GFAPDJ8-BsrGI (SEQ ID NO 25). Several methods for rolling circle amplification were tested, and the best results (high yield and low non-specific amplification) were obtained with the TruePrime technology (Expedeon), which relies on primerless amplification (Picher et al., 2016).

Briefly, the entire column-purified assembly reaction was used in a 900-ul TruePrime reaction following the manufacturer's instructions and incubated overnight at 30° C. The following day, the rolling circle reaction product was incubated 10 minutes at 65° C. to inactivate the enzymes and was diluted 5-fold in 1× thermoPol buffer with 50 ul protelomerase (NEB) in a 4.5-ml reaction. After 1 hour at 30° C., the reaction was heat-treated for 10 minutes at 70° C. to inactivate the protelomerase, and a 4.5-ul aliquot was run on an agarose gel. The entire reaction was then purified on multiple (10-12) Qiagen QiaPrep 2.0 columns following manufacturer's instructions. The typical yield obtained with this method was 160-180 ug DNA, which indicates >100-fold amplification of the starting material (typically 0.5-1 ug) and provides enough DNA for transfection of 200 cell culture dishes (FIG. 16).

The composition of all libraries was tested by next-gen sequencing with an Illumina NextSeq sequencing platform to estimate the number of variants and the eventual contamination by wild-type viruses. Amplicons were generated by PCR with Q5 polymerase (NEB) using primers containing Illumina TruSeq adapters and index barcodes. Amplicons were obtained by low-cycle PCR amplification (15 cycles), ran on 3% agarose gels and purified using Zymo gel extraction reagents. Libraries were quantified using a nanodrop, pooled into equimolar mixes and re-quantified with a KAPA library quantification kit following manufacturer's instruction. Libraries were mixed with 20-40% of PhiX control library to increase sequence diversity.

All DNA libraries generated by rolling circle showed a high sequence diversity (typically >1e8 unique variants, beyond the limits of NextSeq sequencing). By comparison, plasmid libraries generated by bacterial transformation rarely exceeded 1-2e7 variants.

Example 6. Prevention and/or Reduction of Contamination

In another embodiment, a primer/vector system aimed at completely preventing contamination of AAV9 libraries by wild-type virus possibly recovered from environmental contamination or from naturally infected primate animal tissues was created. This was achieved by introducing a maximum number of silent mutations in the sequences surrounding the library insertion site, as well as the sequence immediately before the CAP stop codon, used for PCR amplification (FIG. 17). These libraries showed an extremely low number of wild-type AAV9 detection by NGS (<2 AAV9 reads per 5e7 total reads), suggesting that the alteration of codons surrounding the library amplification and cloning sites is a very efficient way to preserve libraries from environmental or experimental contaminations.

Libraries were produced as described previously by calcium phosphate transfection of HEK-293T cells, dual iodixanol gradient fractionation and membrane ultrafiltration using 100,000 Da MWCO Amicon-15 membranes (Millipore), quantified by real-time PCR and an aliquot was used for NGS amplicon generation and NextSeq sequencing. The diversity of viral libraries was significantly lower than that of DNA libraries (typically ˜1-2e7 unique variants) and showed a very strong counter-selection of variants containing stop codons (from 20% in DNA libraries to ˜1% in virus libraries), evincing a very high rate of cis-packaging, as observed in previous studies (Nonnenmacher et al., 2014).

Example 7. In Vivo Selection of AAV9 Libraries for Mouse Brain Transduction

An RNA-driven library selection for increased brain transduction in a murine model was then developed. AAV9 libraries generated as described above were intravenously injected to male C57BL/6 mice at a dose 2e12 VG per mouse. Two groups of mice were injected with a single SYN-driven or GFAP-driven libraries derived from wild-type AAV9 flanking sequences, and two other groups received pooled libraries containing wild-type and PHP.eB-derived flanking sequences (FIG. 18). After one month, RNA was extracted from 200 mg of brain tissue corresponding to a whole hemisphere using RNeasy Universal Plus procedure (Qiagen). In order to minimize the possibility of RNA under sampling, the entire RNA preparation (˜200 ug) was subjected to mRNA enrichment using Oligotex beads (Qiagen) as recommended by the manufacturer. The entire preparation of enriched mRNA (˜5 ug, equivalent to 2% of total RNA) was then reverse transcribed in a 40-ul Superscript IV reaction (Life Technologies) using a library-specific primer with the following sequence: 5′-GAAACGAATTAAACGGTTTATTGATTAACAATCGATTA-3′ (SEQ ID NO: 415) (CAP stop codon is underlined) (FIG. 19). The entire pool of cDNA was then amplified 30 cycles with 55° C. annealing temperature and 2 minutes elongation in a 500-ul PCR reaction assembled with Q5 master mix, GloSpliceF6 forward primer and a CAP9-specific reverse primer: 5′-CGGTTTATTGATTAACAATCGATTACAGATTACGAGTCAGGTATC-3′ (SEQ ID NO: 416) (CAP stop codon is underlined). This method allowed recovery of abundant amplicons from all brain samples (FIG. 20).

Full-length capsid amplicons were then used as templates for NGS library generation, as well as cloning into a P1 DNA library for the next round of biopanning, using the exact same assembly and cloning-free procedure. NGS analysis performed on PCR amplicons indicated that the library diversity dropped ˜25-fold (from 1e7 to 4e5) after the first round of biopanning for both Syn-driven and GFAP-driven libraries (FIG. 21). The number of 1s^(t) pass variants (P1) recovered was too high to show any significant sequence convergence at this point, and there was very little overlap between the composition of pools recovered from individual animals. Therefore, a second round of selection was performed. After the second biopanning (P2), the total number of unique variants further dropped by 4-5-fold, down to <1e5 peptides. Importantly, some libraries recovered after the first round of biopanning showed significant counts of wild-type AAV9 and AAV-PHP.eB sequences, presumably from environmental contamination. These later became useful benchmarks in the second round of enrichment.

Following RNA recovery and PCR amplification, a systematic enrichment analysis by NGS was performed by calculating the ratio of P2/P1 reads and comparing it to AAV9 or PHP.eB P2/P1 ratio. As shown in FIG. 22, Table 4, FIG. 23 and Table 5, several capsids showed a higher enrichment ratio than the benchmark PHP.eB in both Syn-driven and GFAP-driven libraries, and sequence convergence was obvious, as represented by consensus sequence generation.

TABLE 4 Capsid analysis results Rank SEQ Brain/ (enrichment Ranking ID Average P1 virus factor) (count) Peptide NO of brain AEvirus_S11 stock  1 136 DGTLAVHFK 417    2546.3       6 254.6  2 153 DGTFAVPFK 418    2321.7       6 232.2  3 155 EGTLAVPFK 419    2351.0       7 201.5  4 147 DGTMAVPFK 420    2547.0       8 191.0  5  32 DGTGGTKGW 107   11116.0      35 190.6  6   3 AQWPTSYDA  62  119359.7     512 139.9  7  99 DGTLAVTFK 421    3779.7      19 119.4  8 176 DGTLAVPIK 422    1882.0      13  86.9  9  36 AQTTEKPWL  83   10192.0      76  80.5 10 165 DGTAIHLSS  67    2885.0      23  75.3 11  13 DGTLSQPFR  65   42145.7     344  73.5 12   2 DGTLAAPFK 120  157129.3   1,300  72.5 13   8 AQPEGSARW  60   70884.0     594  71.6 14  48 AQWPTAYDA 256    5934.0      53  67.2 15 198 DGTLQQPFR  89    2793.3      25  67.0 16 104 DGTLAVNFK 346    3511.0      32  65.8 17  31 DGTGNLSGW 302   14521.3     133  65.5 18 158 DGTLEVTFK 423    2337.7      22  63.8 19  51 DGTMDKPFR  70   23962.3     234  61.4 20  80 DGTGQVTGW  68    6242.7      62  60.4 21  42 AQFPTNYDS  66    8640.0      86  60.3 22 127 ERTLAVPFK 424    2873.3      31  55.6 23   1 DGTLAVPFK  71 9885065.7 110,785  53.5 24  61 DGTGTTMGW 324    6753.0      76  53.3 25  69 DGSQSTTGW 136    7227.7      82  52.9 26 186 DGTVSNPFR 403    2074.3      24  51.9 27 160 DGTLEVHFK 348    2245.0      26  51.8 28  29 DGTISQPFK 105   20505.7     243  50.6 29 102 AQGSWNPPA  80    3746.0      45  49.9 30  59 DGTHSTTGW 145    7499.0      91  49.4 31  23 DGTGSTTGW 134   21582.0     272  47.6 32 142 DGTGTTTGW 130    3077.3      39  47.3 33  74 DGTVTTTGW 405    5088.7      66  46.3 34  35 DGTTYVPPR  75    9614.7     126  45.8 35  40 DGTMDRPFK 102    7868.3     104  45.4 36   4 DGTGTTLGW 323   88397.3   1,169  45.4 37 156 DGTALMLSS 280    2444.0      34  43.1 38 116 DGTNTTHGW 113    3065.0      43  42.8 39  98 SGSLAVPFK 425    4107.3      58  42.5 40  38 DGTATTTGW 285   10529.7     150  42.1 41  11 DGTSYVPPR  78   36293.3     526  41.4 42  89 DGTGNTHGW  72    3399.3      50  40.8 43 129 DGTASVTGW 283    4824.3      71  40.8 44  12 AQWELSNGY 246   40837.0     611  40.1 45 115 DGTGNTSGW 137    3405.0      51  40.1 46  67 DGKGSTQGW 272    5818.0      88  39.7 47 137 DGTVIMLSS 397    3781.0      58  39.1 48 119 DGTGGVMGW 297    2302.3      36  38.4 49  58 DGGGTTTGW 270   11174.3     175  38.3 50  71 DGTSIHLSS 378    5703.7      90  38.0

TABLE 5 Capsid analysis results Rank SEQ Brain/ (enrichment Ranking ID Average p1 virus factor) (count) Peptide NO of brain AEvirus_S11 stock  1 106 DGTGGTKGW 107    3620.7    0 NA  2 264 GGTRNTAPM 426     831.0    0 NA  3 295 AQGRMTDSQ 199     716.0    0 NA  4 677 DGNSYVPPR 427     474.3    0 NA  5 700 AQAGVSGQR 428     456.0    0 NA  6 731 AQAGNSNAV 429     844.0    0 NA  7 181 DGTGGLTGW 294    4044.3    4 606.7  8 558 AQWVYGQTV 430     977.7    1 586.6  9 123 DGTSFSPPK 431    4227.3   10 253.6 10  35 DGTIERPFR  87   29872.0   92 194.8 11 105 DGTTLVPPR 116    5597.3   19 176.8 12  18 DGTADKPFR  63  103305.3  363 170.8 13  22 DGTASYYDS  61   61841.3  233 159.2 14  26 AQTTDRPFL  85   38893.7  147 158.7 15   8 DGTQFSPPR 108  206660.7  801 154.8 16 169 DGTTTYGAR  77    4237.3   17 149.6 17  11 AQFVVGQQY  95  152965.0  625 146.8 18  61 DGTSYVPPR  78   13968.0   58 144.5 19  16 DGTAERPFR 140  134132.7  565 142.4 20  21 AQGENPGRW  96   68919.7  292 141.6 21 157 DGTSFTPPR  88    3210.0   14 137.6 22  73 AQTLARPFV  98    5947.7   26 137.3 23   9 DGTTWTPPR 139  184936.7  825 134.5 24 721 DGTATTMGW 284    5562.3   25 133.5 25 129 AQGTWNPPA  82   12379.3   57 130.3 26 215 DGTRLMLSS 368    2505.0   12 125.3 27  60 AQPLAVYGA 217   13419.3   66 122.0 28 909 AQGLDLGRW 432     405.0    2 121.5 29  53 DGTSFTPPK  81   13673.3   68 120.6 30 412 AQVMSGVGQ 433     583.0    3 116.6 31 390 AQKSVGSVY 205    4415.7   23 115.2 32  70 AQTREYLLG  93    5752.7   30 115.1 33  43 DGTNGLKGW  76   15068.7   79 114.4 34  93 AQYLAGYTV 262    6223.3   33 113.2 35  54 AQTGFAPPR 161   14611.3   78 112.4 36 115 DGTLNNPFR 109    4719.7   26 108.9 37 968 DGNGGLKGW 167    3199.0   18 106.6 38 120 AQSVAKPFL 231    6929.7   39 106.6 39 544 DGTHGLRGW 434     528.0    3 105.6 40 159 AQSVVRPFL 233    2457.3   14 105.3 41  65 DGTRNMYEG 135   21086.3  124 102.0 42 556 AQRWAADSS 435     500.7    3 100.1 43  30 AQGPTRPFL 125   46225.3  279  99.4 44  64 DGTVPYLSS 401   22384.3  137  98.0 45 870 AQTGASGAT 436     473.7    3  94.7 46 341 AQLVAGYSQ 437    1240.0    8  93.0 47 375 AQSGGVGQV 228     768.3    5  92.2 48 145 AQSLARLFP 438    4435.3   29  91.8 49   1 DGTLAVPFK  71 1445517.0 9453  91.7 50 124 DGTGNVTGW  69    5424.3   36  90.4

Importantly, there was also a strong sequence convergence between different animals, suggesting an efficient selection after only two passages. FIG. 24 and FIG. 25 provide an estimation of brain/liver specificity in GFAP-AAV9 peptide library candidates.

Example 8. Multiplexing Selections

For the final multiplex in vivo screen by individual variant pooling in equimolar library, a subpopulation of variants with promising properties (such as, but not limited to, enrichment factor, liver detargeting, high counts in more than one mouse, etc.) may be selected as shown in FIG. 26 and then an equimolar pool of primers encoding all the 7-mers (microchip solid-phase synthesis, up to 3,800 primers per chip) can be synthesized. The limited diversity library may be produced including internal controls such as, but not limited to, PHP.N, PHP.B, wild-type AAV9 (wtAAV9) and/or any other serotype including those taught herein. The mice are injected and then the RNA enrichment is compared to internal controls in a similar manner to a barcoding study, which is known in the art and described herein.

Example 9. Codon Optimization

Codon variants may be used to improve data strength when using synthesized libraries. A listing of NNK codons, NNM codons and the most favorable NNM codons in mammals for various amino acids is provided in Table 6. In Table 6, * means that no NNM codon was available and ** means “avoid homopolymeric stretches if possible.”

TABLE 6 Codon Variants Most favorable NNM Amino NN K NN M codon in acid codon codons mammals F TTT TTC TTC L TTG, CTT, CTG TTA, CTC, CTA CTC S TCT, TCG, AGT TCC, TCA, AGC AGC Y TAT TAC TAC C TGT TGC TGC W TGG TGG* P CCT, CCG CCC, CCA CCA** H CAT CAC CAC Q CAG CAA CAA R CGT, CGG, AGG CGC, CGA, AGA AGA I ATT ATC, ATA ATC M ATG ATT* T ACT, ACG ACC, ACA ACC N AAT AAC AAC K AAG AAA AAA V GTT, GTG GTC, GTA GTC A GCT, GCG GCC, GCA GCC D GAT GAC GAC E GAG GAA GAA G GGT, GGG GGC, GGA GGC stop TAG TAC, TAA n/a *no NNM codon available **avoid homopolymeric stretches if possible

In order to have a balanced library it is recommended to establish a list of potential candidates. Then, using Table 6, a pooled primer library containing every peptide variant with encoded by NNK codons (original from library) and non-NNK codons (maximum variation). If similar behavior is seen between the two variants of the same peptide, this would strengthen the analysis of that peptide. Additionally, it is recommended to choose the most favorable NNM codons (M=A or C).

Example 10. Library Generation

The top-ranking 330 peptide variants from SYN-driven and GFAP-driven libraries that showed enhanced performance relative to the parental AAV9 were selected. A de novo library by pooled primer synthesis of all 330 peptide sequences plus AAV9, AAV-PHP.B and AAV-PHP.eB controls was generated (Table 7). In order to exclude potential artifacts due to the DNA sequence and to increase the robustness of the assay, each peptide variant was encoded by two different DNA sequences, one where all amino acids were encoded by NNK codons (identical to the original library) and another one where NNM codons were used whenever possible (M=C or A, Table 6).

TABLE 7 Peptide variants selected after 2 rounds of RNA-driven mouse brain biopanning SEQ Nucleotide SEQ Nucleotide SEQ Peptide ID sequence ID sequence ID Sequence NO: (NNK codons) NO: (NNM codons) NO: AQ (AAV9) CAGAGTGCTCAG 439 CAGAGTGCCCAA  772 GCACAG GCACAG AQAGAGSER 194 CAGAGTGCCCAA 440 CAGAGTGCACAA  773 GCGGGTGCGGGG GCAGGAGCAGGA TCGGAGCGGGCA AGCGAAAGAGCA CAG CAG AQDQNPGRW 195 CAGAGTGCCCAA 441 CAGAGTGCACAA  774 GATCAGAATCCG GACCAAAACCCA GGGCGTTGGGCA GGAAGATGGGCA CAG CAG AQELTRPFL 144 CAGAGTGCCCAA 442 CAGAGTGCACAA  775 GAGTTGACGCGT GAACTCACAAGA CCGTTTTTGGCAC CCATTCCTCGCAC AG AG AQEVPGYRW 196 CAGAGTGCCCAA 443 CAGAGTGCACAA  776 GAGGTGCCTGGG GAAGTCCCAGGA TATAGGTGGGCA TACAGATGGGCA CAG CAG AQFPTNYDS  66 CAGAGTGCCCAA 444 CAGAGTGCACAA  777 TTTCCTACGAATT TTCCCAACAAACT ATGATTCTGCACA ACGACAGCGCAC G AG AQFVVGQQY  95 CAGAGTGCCCAA 445 CAGAGTGCACAA  778 TTTGTGGTTGGTC TTCGTCGTCGGAC AGCAGTATGCAC AACAATACGCAC AG AG AQGASPGRW 149 CAGAGTGCCCAA 446 CAGAGTGCACAA  779 GGGGCTAGTCCG GGAGCAAGCCCA GGGCGGTGGGCA GGAAGATGGGCA CAG CAG AQGENPGRW  96 CAGAGTGCCCAA 447 CAGAGTGCACAA  780 GGGGAGAATCCG GGAGAAAACCCA GGTAGGTGGGCA GGAAGATGGGCA CAG CAG AQGGNPGRW  91 CAGAGTGCCCAA 448 CAGAGTGCACAA  781 GGGGGGAATCCG GGAGGAAACCCA GGTCGGTGGGCA GGAAGATGGGCA CAG CAG AQGGSTGSN 197 CAGAGTGCCCAA 449 CAGAGTGCACAA  782 GGTGGTTCTACG GGAGGAAGCACA GGGTCGAATGCA GGAAGCAACGCA CAG CAG AQGPTRPFL 125 CAGAGTGCCCAA 450 CAGAGTGCACAA  783 GGGCCGACTAGG GGACCAACAAGA CCGTTTTTGGCAC CCATTCCTCGCAC AG AG AQGRDGWAA 198 CAGAGTGCCCAA 451 CAGAGTGCACAA  784 GGTCGGGATGGT GGAAGAGACGGA TGGGCGGCGGCA TGGGCAGCAGCA CAG CAG AQGRMTDSQ 199 CAGAGTGCCCAA 452 CAGAGTGCACAA  785 GGTCGTATGACT GGAAGAATGACA GATTCGCAGGCA GACAGCCAAGCA CAG CAG AQGSDVGRW 128 CAGAGTGCCCAA 453 CAGAGTGCACAA  786 GGTAGTGATGTG GGAAGCGACGTC GGGCGGTGGGCA GGAAGATGGGCA CAG CAG AQGSNPGRW 103 CAGAGTGCCCAA 454 CAGAGTGCACAA  787 GGTAGTAATCCG GGAAGCAACCCA GGGAGGTGGGCA GGAAGATGGGCA CAG CAG AQGSNSPQV 200 CAGAGTGCCCAA 455 CAGAGTGCACAA  788 GGGTCTAATTCGC GGAAGCAACAGC CTCAGGTGGCAC CCACAAGTCGCA AG CAG AQGSWNPPA  80 CAGAGTGCCCAA 456 CAGAGTGCACAA  789 GGTTCGTGGAAT GGAAGCTGGAAC CCGCCGGCGGCA CCACCAGCAGCA CAG CAG AQGTWNPPA  82 CAGAGTGCCCAA 457 CAGAGTGCACAA  790 GGTACTTGGAAT GGAACATGGAAC CCGCCGGCTGCA CCACCAGCAGCA CAG CAG AQGVFIPPK 201 CAGAGTGCCCAA 458 CAGAGTGCACAA  791 GGTGTTTTTATTC GGAGTCTTCATCC CGCCGAAGGCAC CACCAAAAGCAC AG AG AQHVNASQS 202 CAGAGTGCCCAA 459 CAGAGTGCACAA  792 CATGTGAATGCTT CACGTCAACGCA CTCAGTCTGCACA AGCCAAAGCGCA G CAG AQIKAGWAQ 203 CAGAGTGCCCAA 460 CAGAGTGCACAA  793 ATTAAGGCGGGG ATCAAAGCAGGA TGGGCGCAGGCA TGGGCACAAGCA CAG CAG AQIMSGYAQ 204 CAGAGTGCCCAA 461 CAGAGTGCACAA  794 ATTATGAGTGGG ATCATGAGCGGA TATGCTCAGGCA TACGCACAAGCA CAG CAG AQKSVGSVY 205 CAGAGTGCCCAA 462 CAGAGTGCACAA  795 AAGAGTGTGGGT AAAAGCGTCGGA AGTGTTTATGCAC AGCGTCTACGCA AG CAG AQLEHGFAQ 206 CAGAGTGCCCAA 463 CAGAGTGCACAA  796 CTTGAGCATGGG CTCGAACACGGA TTTGCTCAGGCAC TTCGCACAAGCA AG CAG AQLGGVLSA 207 CAGAGTGCCCAA 464 CAGAGTGCACAA  797 CTGGGTGGGGTG CTCGGAGGAGTC TTGAGTGCTGCAC CTCAGCGCAGCA AG CAG AQLGLSQGR 208 CAGAGTGCCCAA 465 CAGAGTGCACAA  798 CTGGGGCTTTCGC CTCGGACTCAGC AGGGGCGGGCAC CAAGGAAGAGCA AG CAG AQLGYGFAQ 209 CAGAGTGCCCAA 466 CAGAGTGCACAA  799 TTGGGGTATGGG CTCGGATACGGA TTTGCTCAGGCAC TTCGCACAAGCA AG CAG AQLKYGLAQ 115 CAGAGTGCCCAA 467 CAGAGTGCACAA  800 TTGAAGTATGGTC CTCAAATACGGA TTGCGCAGGCAC CTCGCACAAGCA AG CAG AQLRIGFAQ 210 CAGAGTGCCCAA 468 CAGAGTGCACAA  801 CTTCGGATTGGTT CTCAGAATCGGA TTGCTCAGGCAC TTCGCACAAGCA AG CAG AQLRMGYSQ 211 CAGAGTGCCCAA 469 CAGAGTGCACAA  802 TTGCGTATGGGTT CTCAGAATGGGA ATAGTCAGGCAC TACAGCCAAGCA AG CAG AQLRQGYAQ 212 CAGAGTGCCCAA 470 CAGAGTGCACAA  803 CTGAGGCAGGGG CTCAGACAAGGA TATGCTCAGGCA TACGCACAAGCA CAG CAG AQLRVGFAQ 123 CAGAGTGCCCAA 471 CAGAGTGCACAA  804 TTGCGTGTTGGTT CTCAGAGTCGGA TTGCGCAGGCAC TTCGCACAAGCA AG CAG AQLSCRSQM 213 CAGAGTGCCCAA 472 CAGAGTGCACAA  805 CTGTCGTGTCGGA CTCAGCTGCAGA GTCAGATGGCAC AGCCAAATGGCA AG CAG AQLTYSQSL 214 CAGAGTGCCCAA 473 CAGAGTGCACAA  806 TTGACGTATAGTC CTCACATACAGC AGTCGCTGGCAC CAAAGCCTCGCA AG CAG AQLYKGYSQ 215 CAGAGTGCCCAA 474 CAGAGTGCACAA  807 CTGTATAAGGGTT CTCTACAAAGGA ATAGTCAGGCAC TACAGCCAAGCA AG CAG AQMPQRPFL 216 CAGAGTGCCCAA 475 CAGAGTGCACAA  808 ATGCCTCAGCGG ATGCCACAAAGA CCGTTTTTGGCAC CCATTCCTCGCAC AG AG AQNGNPGRW  84 CAGAGTGCCCAA 476 CAGAGTGCACAA  809 AATGGTAATCCG AACGGAAACCCA GGGCGGTGGGCA GGAAGATGGGCA CAG CAG AQPEGSARW  60 CAGAGTGCCCAA 477 CAGAGTGCACAA  810 CCTGAGGGTAGT CCAGAAGGAAGC GCGAGGTGGGCA GCAAGATGGGCA CAG CAG AQPLAVYGA 217 CAGAGTGCCCAA 478 CAGAGTGCACAA  811 CCGTTGGCTGTTT CCACTCGCAGTCT ATGGGGCGGCAC ACGGAGCAGCAC AG AG AQPQSSSMS 218 CAGAGTGCCCAA 479 CAGAGTGCACAA  812 CCGCAGTCGTCGT CCACAAAGCAGC CGATGAGTGCAC AGCATGAGCGCA AG CAG AQPSVGGYW 219 CAGAGTGCCCAA 480 CAGAGTGCACAA  813 CCGAGTGTGGGT CCAAGCGTCGGA GGGTATTGGGCA GGATACTGGGCA CAG CAG AQQAVGQSW 220 CAGAGTGCCCAA 481 CAGAGTGCACAA  814 CAGGCTGTGGGT CAAGCAGTCGGA CAGTCTTGGGCA CAAAGCTGGGCA CAG CAG AQQRSLASG 221 CAGAGTGCCCAA 482 CAGAGTGCACAA  815 CAGCGTTCGCTG CAAAGAAGCCTC GCTTCGGGTGCA GCAAGCGGAGCA CAG CAG AQQVMNSQG 222 CAGAGTGCCCAA 483 CAGAGTGCACAA  816 CAGGTGATGAAT CAAGTCATGAAC AGTCAGGGGGCA AGCCAAGGAGCA CAG CAG AQRGVGLSQ 223 CAGAGTGCCCAA 484 CAGAGTGCACAA  817 CGTGGGGTTGGG AGAGGAGTCGGA TTGAGTCAGGCA CTCAGCCAAGCA CAG CAG AQRHDAEGS 224 CAGAGTGCCCAA 485 CAGAGTGCACAA  818 AGGCATGATGCG AGACACGACGCA GAGGGTAGTGCA GAAGGAAGCGCA CAG CAG AQRKGEPHY 225 CAGAGTGCCCAA 486 CAGAGTGCACAA  819 CGTAAGGGGGAG AGAAAAGGAGAA CCTCATTATGCAC CCACACTACGCA AG CAG AQRYTGDSS 138 CAGAGTGCCCAA 487 CAGAGTGCACAA  820 AGGTATACGGGG AGATACACAGGA GATTCTAGTGCAC GACAGCAGCGCA AG CAG AQSAMAAKG 226 CAGAGTGCCCAA 488 CAGAGTGCACAA  821 TCGGCGATGGCT AGCGCAATGGCA GCGAAGGGTGCA GCAAAAGGAGCA CAG CAG AQSGGLTGS 227 CAGAGTGCCCAA 489 CAGAGTGCACAA  822 TCTGGGGGTCTTA AGCGGAGGACTC CGGGGAGTGCAC ACAGGAAGCGCA AG CAG AQSGGVGQV 228 CAGAGTGCCCAA 490 CAGAGTGCACAA  823 TCGGGTGGGGTG AGCGGAGGAGTC GGGCAGGTGGCA GGACAAGTCGCA CAG CAG AQSLATPFR 169 CAGAGTGCCCAA 491 CAGAGTGCACAA  824 TCTCTGGCGACGC AGCCTCGCAACA CTTTTCGTGCACA CCATTCAGAGCA G CAG AQSMSRPFL 229 CAGAGTGCCCAA 492 CAGAGTGCACAA  825 AGTATGTCGCGTC AGCATGAGCAGA CGTTTCTGGCACA CCATTCCTCGCAC G AG AQSQLRPFL 230 CAGAGTGCCCAA 493 CAGAGTGCACAA  826 AGTCAGCTTAGG AGCCAACTCAGA CCGTTTCTTGCAC CCATTCCTCGCAC AG AG AQSVAKPFL 231 CAGAGTGCCCAA 494 CAGAGTGCACAA  827 TCTGTGGCTAAGC AGCGTCGCAAAA CTTTTTTGGCACA CCATTCCTCGCAC G AG AQSVSQPFR 232 CAGAGTGCCCAA 495 CAGAGTGCACAA  828 TCGGTTTCGCAGC AGCGTCAGCCAA CGTTTAGGGCAC CCATTCAGAGCA AG CAG AQSVVRPFL 233 CAGAGTGCCCAA 496 CAGAGTGCACAA  829 TCTGTGGTGCGTC AGCGTCGTCAGA CTTTTCTGGCACA CCATTCCTCGCAC G AG AQTALSSST 234 CAGAGTGCCCAA 497 CAGAGTGCACAA  830 ACTGCGCTTTCGT ACAGCACTCAGC CGTCGACGGCAC AGCAGCACAGCA AG CAG AQTEMGGRC 235 CAGAGTGCCCAA 498 CAGAGTGCACAA  831 ACGGAGATGGGT ACAGAAATGGGA GGGAGGTGTGCA GGAAGATGCGCA CAG CAG AQTGFAPPR 161 CAGAGTGCCCAA 499 CAGAGTGCACAA  832 ACGGGGTTTGCTC ACAGGATTCGCA CGCCGCGTGCAC CCACCAAGAGCA AG CAG AQTIRGYSS 236 CAGAGTGCCCAA 500 CAGAGTGCACAA  833 ACGATTCGGGGG ACAATCAGAGGA TATTCGTCTGCAC TACAGCAGCGCA AG CAG AQTISNYHT 237 CAGAGTGCCCAA 501 CAGAGTGCACAA  834 ACTATTTCTAATT ACAATCAGCAAC ATCATACGGCAC TACCACACAGCA AG CAG AQTLARPFV  98 CAGAGTGCCCAA 502 CAGAGTGCACAA  835 ACTTTGGCGCGTC ACACTCGCAAGA CGTTTGTGGCACA CCATTCGTCGCAC G AG AQTLAVPFK 168 CAGAGTGCCCAA 503 CAGAGTGCACAA  836 (PHP.B) ACTTTGGCGGTGC ACACTCGCAGTC CTTTTAAGGCACA CCATTCAAAGCA G CAG AQTPDRPWL 238 CAGAGTGCCCAA 504 CAGAGTGCACAA  837 ACTCCTGATCGTC ACACCAGACAGA CTTGGTTGGCACA CCATGGCTCGCA G CAG AQTRAGYAQ 126 CAGAGTGCCCAA 505 CAGAGTGCACAA  838 ACTCGGGCTGGG ACAAGAGCAGGA TATGCTCAGGCA TACGCACAAGCA CAG CAG AQTRAGYSQ 141 CAGAGTGCCCAA 506 CAGAGTGCACAA  839 ACTAGGGCGGGG ACAAGAGCAGGA TATTCTCAGGCAC TACAGCCAAGCA AG CAG AQTREYLLG  93 CAGAGTGCCCAA 507 CAGAGTGCACAA  840 ACGCGTGAGTAT ACAAGAGAATAC CTGCTGGGGGCA CTCCTCGGAGCA CAG CAG AQTSAKPFL 163 CAGAGTGCCCAA 508 CAGAGTGCACAA  841 ACTTCTGCGAAG ACAAGCGCAAAA CCGTTTCTTGCAC CCATTCCTCGCAC AG AG AQTSARPFL 100 CAGAGTGCCCAA 509 CAGAGTGCACAA  842 ACTTCTGCTAGGC ACAAGCGCAAGA CTTTTCTGGCACA CCATTCCTCGCAC G AG AQTTDRPFL  85 CAGAGTGCCCAA 510 CAGAGTGCACAA  843 ACTACTGATAGG ACAACAGACAGA CCTTTTTTGGCAC CCATTCCTCGCAC AG AG AQTTEKPWL  83 CAGAGTGCCCAA 511 CAGAGTGCACAA  844 ACGACTGAGAAG ACAACAGAAAAA CCGTGGCTGGCA CCATGGCTCGCA CAG CAG AQTVARPFY 239 CAGAGTGCCCAA 512 CAGAGTGCACAA  845 ACGGTTGCGCGG ACAGTCGCAAGA CCTTTTTATGCAC CCATTCTACGCAC AG AG AQTVATPFR 240 CAGAGTGCCCAA 513 CAGAGTGCACAA  846 ACTGTTGCTACGC ACAGTCGCAACA CGTTTAGGGCAC CCATTCAGAGCA AG CAG AQTVTQLFK 241 CAGAGTGCCCAA 514 CAGAGTGCACAA  847 ACGGTGACGCAG ACAGTCACACAA TTGTTTAAGGCAC CTCTTCAAAGCAC AG AG AQVHVGSVY 165 CAGAGTGCCCAA 515 CAGAGTGCACAA  848 GTTCATGTTGGGA GTCCACGTCGGA GTGTTTATGCACA AGCGTCTACGCA G CAG AQVLAGYNM 242 CAGAGTGCCCAA 516 CAGAGTGCACAA  849 GTTCTTGCTGGGT GTCCTCGCAGGA ATAATATGGCAC TACAACATGGCA AG CAG AQVSEARVR 243 CAGAGTGCCCAA 517 CAGAGTGCACAA  850 GTTTCTGAGGCG GTCAGCGAAGCA AGGGTTAGGGCA AGAGTCAGAGCA CAG CAG AQVVVGYSQ 244 CAGAGTGCCCAA 518 CAGAGTGCACAA  851 GTTGTGGTGGGTT GTCGTCGTCGGAT ATAGTCAGGCAC ACAGCCAAGCAC AG AG AQWAAGYNV 245 CAGAGTGCCCAA 519 CAGAGTGCACAA  852 TGGGCTGCTGGG TGGGCAGCAGGA TATAATGTGGCA TACAACGTCGCA CAG CAG AQWELSNGY 246 CAGAGTGCCCAA 520 CAGAGTGCACAA  853 TGGGAGCTGAGT TGGGAACTCAGC AATGGGTATGCA AACGGATACGCA CAG CAG AQWEVKGGY 247 CAGAGTGCCCAA 521 CAGAGTGCACAA  854 TGGGAGGTGAAG TGGGAAGTCAAA GGGGGTTATGCA GGAGGATACGCA CAG CAG AQWEVKRGY 248 CAGAGTGCCCAA 522 CAGAGTGCACAA  855 TGGGAGGTGAAG TGGGAAGTCAAA CGGGGGTATGCA AGAGGATACGCA CAG CAG AQWEVQSGF 249 CAGAGTGCCCAA 523 CAGAGTGCACAA  856 TGGGAGGTTCAG TGGGAAGTCCAA TCTGGGTTTGCAC AGCGGATTCGCA AG CAG AQWEVRGGY 250 CAGAGTGCCCAA 524 CAGAGTGCACAA  857 TGGGAGGTTCGT TGGGAAGTCAGA GGTGGTTATGCA GGAGGATACGCA CAG CAG AQWEVTSGW 251 CAGAGTGCCCAA 525 CAGAGTGCACAA  858 TGGGAGGTGACG TGGGAAGTCACA AGTGGTTGGGCA AGCGGATGGGCA CAG CAG AQWGAPSHG 252 CAGAGTGCCCAA 526 CAGAGTGCACAA  859 TGGGGGGCGCCG TGGGGAGCACCA AGTCATGGGGCA AGCCACGGAGCA CAG CAG AQWMELGSS 253 CAGAGTGCCCAA 527 CAGAGTGCACAA  860 TGGATGGAGCTT TGGATGGAACTC GGTAGTTCGGCA GGAAGCAGCGCA CAG CAG AQWMFGGSG 254 CAGAGTGCCCAA 528 CAGAGTGCACAA  861 TGGATGTTTGGG TGGATGTTCGGA GGTAGTGGGGCA GGAAGCGGAGCA CAG CAG AQWMLGGAQ 255 CAGAGTGCCCAA 529 CAGAGTGCACAA  862 TGGATGCTGGGG TGGATGCTCGGA GGGGCGCAGGCA GGAGCACAAGCA CAG CAG AQWPTAYDA 256 CAGAGTGCCCAA 530 CAGAGTGCACAA  863 TGGCCGACTGCTT TGGCCAACAGCA ATGATGCGGCAC TACGACGCAGCA AG CAG AQWPTSYDA  62 CAGAGTGCCCAA 531 CAGAGTGCACAA  864 TGGCCTACGAGTT TGGCCAACAAGC ATGATGCTGCAC TACGACGCAGCA AG CAG AQWQVQTGF 257 CAGAGTGCCCAA 532 CAGAGTGCACAA  865 TGGCAGGTTCAG TGGCAAGTCCAA ACGGGGTTTGCA ACAGGATTCGCA CAG CAG AQWSTEGGY 258 CAGAGTGCCCAA 533 CAGAGTGCACAA  866 TGGTCGACTGAG TGGAGCACAGAA GGTGGGTATGCA GGAGGATACGCA CAG CAG AQWTAAGGY 259 CAGAGTGCCCAA 534 CAGAGTGCACAA  867 TGGACTGCTGCG TGGACAGCAGCA GGTGGTTATGCA GGAGGATACGCA CAG CAG AQWTTESGY 260 CAGAGTGCCCAA 535 CAGAGTGCACAA  868 TGGACGACGGAG TGGACAACAGAA TCGGGTTATGCAC AGCGGATACGCA AG CAG AQWVYGSSH 261 CAGAGTGCCCAA 536 CAGAGTGCACAA  869 TGGGTTTATGGG TGGGTCTACGGA AGTTCGCATGCA AGCAGCCACGCA CAG CAG AQYLAGYTV 262 CAGAGTGCCCAA 537 CAGAGTGCACAA  870 TATTTGGCGGGGT TACCTCGCAGGA ATACGGTGGCAC TACACAGTCGCA AG CAG AQYLKGYSV 152 CAGAGTGCCCAA 538 CAGAGTGCACAA  871 TATCTGAAGGGG TACCTCAAAGGA TATTCTGTGGCAC TACAGCGTCGCA AG CAG AQYLSGYNT 263 CAGAGTGCCCAA 539 CAGAGTGCACAA  872 TATTTGTCGGGTT TACCTCAGCGGA ATAATACGGCAC TACAACACAGCA AG CAG DGAAATTGW 264 CAGAGTGATGGC 540 CAGAGTGACGGA  873 GCTGCGGCGACT GCAGCAGCAACA ACTGGGTGGGCA ACAGGATGGGCA CAG CAG DGAGGTSGW 151 CAGAGTGATGGC 541 CAGAGTGACGGA  874 GCGGGTGGGACG GCAGGAGGAACA AGTGGTTGGGCA AGCGGATGGGCA CAG CAG DGAGTTSGW 265 CAGAGTGATGGC 542 CAGAGTGACGGA  875 GCGGGTACTACTT GCAGGAACAACA CGGGTTGGGCAC AGCGGATGGGCA AG CAG DGAHGLSGW 266 CAGAGTGATGGC 543 CAGAGTGACGGA  876 GCTCATGGGCTGT GCACACGGACTC CGGGGTGGGCAC AGCGGATGGGCA AG CAG DGAHVGLSS 267 CAGAGTGATGGC 544 CAGAGTGACGGA  877 GCTCATGTTGGGC GCACACGTCGGA TGTCGTCGGCAC CTCAGCAGCGCA AG CAG DGARTVLQL 268 CAGAGTGATGGC 545 CAGAGTGACGGA  878 GCTCGGACGGTG GCAAGAACAGTC CTTCAGTTGGCAC CTCCAACTCGCAC AG AG DGEYQKPFR 269 CAGAGTGATGGC 546 CAGAGTGACGGA  879 GAGTATCAGAAG GAATACCAAAAA CCGTTTAGGGCA CCATTCAGAGCA CAG CAG DGGGTTTGW 270 CAGAGTGATGGC 547 CAGAGTGACGGA  880 GGTGGGACTACG GGAGGAACAACA ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGHATSMGW 271 CAGAGTGATGGC 548 CAGAGTGACGGA  881 CATGCGACGAGT CACGCAACAAGC ATGGGTTGGGCA ATGGGATGGGCA CAG CAG DGKGSTQGW 272 CAGAGTGATGGC 549 CAGAGTGACGGA  882 AAGGGTTCGACG AAAGGAAGCACA CAGGGGTGGGCA CAAGGATGGGCA CAG CAG DGKQYQLSS  92 CAGAGTGATGGC 550 CAGAGTGACGGA  883 AAGCAGTATCAG AAACAATACCAA CTGTCTTCGGCAC CTCAGCAGCGCA AG CAG DGNGGLKGW 167 CAGAGTGATGGC 551 CAGAGTGACGGA  884 AATGGTGGGTTG AACGGAGGACTC AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGQGGLSGW 273 CAGAGTGATGGC 552 CAGAGTGACGGA  885 CAGGGGGGTTTG CAAGGAGGACTC TCTGGGTGGGCA AGCGGATGGGCA CAG CAG DGQHFAPPR 110 CAGAGTGATGGC 553 CAGAGTGACGGA  886 CAGCATTTTGCTC CAACACTTCGCA CGCCGCGGGCAC CCACCAAGAGCA AG CAG DGRATKTLY 274 CAGAGTGATGGC 554 CAGAGTGACGGA  887 CGTGCGACTAAG AGAGCAACAAAA ACGCTTTATGCAC ACACTCTACGCA AG CAG DGRNALTGW 275 CAGAGTGATGGC 555 CAGAGTGACGGA  888 CGTAATGCGTTG AGAAACGCACTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGRRQVIQL 276 CAGAGTGATGGC 556 CAGAGTGACGGA  889 AGGAGGCAGGTG AGAAGACAAGTC ATTCAGCTGGCA ATCCAACTCGCA CAG CAG DGRVYGLSS 277 CAGAGTGATGGC 557 CAGAGTGACGGA  890 AGGGTTTATGGTC AGAGTCTACGGA TTTCGTCGGCACA CTCAGCAGCGCA G CAG DGSGRTTGW 147 CAGAGTGATGGC 558 CAGAGTGACGGA  891 AGTGGGCGTACG AGCGGAAGAACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGSGTTRGW 114 CAGAGTGATGGC 559 CAGAGTGACGGA  892 TCTGGTACGACG AGCGGAACAACA CGGGGTTGGGCA AGAGGATGGGCA CAG CAG DGSGTVSGW 278 CAGAGTGATGGC 560 CAGAGTGACGGA  893 TCGGGTACGGTT AGCGGAACAGTC AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGSPEKPFR 160 CAGAGTGATGGC 561 CAGAGTGACGGA  894 AGTCCGGAGAAG AGCCCAGAAAAA CCGTTTCGGGCAC CCATTCAGAGCA AG CAG DGSQSTTGW 136 CAGAGTGATGGC 562 CAGAGTGACGGA  895 AGTCAGTCTACTA AGCCAAAGCACA CGGGGTGGGCAC ACAGGATGGGCA AG CAG DGSSFYPPK 127 CAGAGTGATGGC 563 CAGAGTGACGGA  896 AGTAGTTTTTATC AGCAGCTTCTACC CTCCTAAGGCAC CACCAAAAGCAC AG AG DGSSSYYDA  64 CAGAGTGATGGC 564 CAGAGTGACGGA  897 AGTAGTTCTTATT AGCAGCAGCTAC ATGATGCGGCAC TACGACGCAGCA AG CAG DGSTERPFR  99 CAGAGTGATGGC 565 CAGAGTGACGGA  898 TCTACGGAGAGG AGCACAGAAAGA CCGTTTAGGGCA CCATTCAGAGCA CAG CAG DGTAARLSS 132 CAGAGTGATGGC 566 CAGAGTGACGGA  899 ACCGCGGCTCGG ACAGCAGCAAGA CTGTCGTCGGCAC CTCAGCAGCGCA AG CAG DGTADKPFR  63 CAGAGTGATGGC 567 CAGAGTGACGGA  900 ACCGCTGATAAG ACAGCAGACAAA CCGTTTCGGGCAC CCATTCAGAGCA AG CAG DGTADRPFR 155 CAGAGTGATGGC 568 CAGAGTGACGGA  901 ACGGCGGATCGT ACAGCAGACAGA CCTTTTCGGGCAC CCATTCAGAGCA AG CAG DGTAERPFR 140 CAGAGTGATGGC 569 CAGAGTGACGGA  902 ACCGCGGAGAGG ACAGCAGAAAGA CCTTTTAGGGCAC CCATTCAGAGCA AG CAG DGTAIHLSS  67 CAGAGTGATGGC 570 CAGAGTGACGGA  903 ACCGCGATTCATC ACAGCAATCCAC TTTCGTCTGCACA CTCAGCAGCGCA G CAG DGTAIYLSS 279 CAGAGTGATGGC 571 CAGAGTGACGGA  904 ACCGCGATTTATC ACAGCAATCTAC TGTCTTCTGCACA CTCAGCAGCGCA G CAG DGTALMLSS 280 CAGAGTGATGGC 572 CAGAGTGACGGA  905 ACCGCTCTTATGT ACAGCACTCATG TGTCGTCTGCACA CTCAGCAGCGCA G CAG DGTASISGW 281 CAGAGTGATGGC 573 CAGAGTGACGGA  906 ACCGCGAGTATT ACAGCAAGCATC AGTGGTTGGGCA AGCGGATGGGCA CAG CAG DGTASTSGW 282 CAGAGTGATGGC 574 CAGAGTGACGGA  907 ACCGCGTCGACG ACAGCAAGCACA AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGTASVTGW 283 CAGAGTGATGGC 575 CAGAGTGACGGA  908 ACCGCGTCGGTG ACAGCAAGCGTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTASYYDS  61 CAGAGTGATGGC 576 CAGAGTGACGGA  909 ACCGCGAGTTATT ACAGCAAGCTAC ATGATTCTGCACA TACGACAGCGCA G CAG DGTATTMGW 284 CAGAGTGATGGC 577 CAGAGTGACGGA  910 ACCGCGACGACG ACAGCAACAACA ATGGGGTGGGCA ATGGGATGGGCA CAG CAG DGTATTTGW 285 CAGAGTGATGGC 578 CAGAGTGACGGA  911 ACCGCGACGACG ACAGCAACAACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTAYRLSS 286 CAGAGTGATGGC 579 CAGAGTGACGGA  912 ACCGCGTATCGTT ACAGCATACAGA TGTCGTCTGCACA CTCAGCAGCGCA G CAG DGTDKMWSI 287 CAGAGTGATGGC 580 CAGAGTGACGGA  913 ACCGATAAGATG ACAGACAAAATG TGGAGTATTGCA TGGAGCATCGCA CAG CAG DGTGGIKGW 131 CAGAGTGATGGC 581 CAGAGTGACGGA  914 ACCGGTGGTATT ACAGGAGGAATC AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGTGGIMGW 288 CAGAGTGATGGC 582 CAGAGTGACGGA  915 ACCGGGGGGATT ACAGGAGGAATC ATGGGTTGGGCA ATGGGATGGGCA CAG CAG DGTGGISGW 289 CAGAGTGATGGC 583 CAGAGTGACGGA  916 ACCGGTGGGATT ACAGGAGGAATC TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGGLAGW 290 CAGAGTGATGGC 584 CAGAGTGACGGA  917 ACCGGGGGTCTT ACAGGAGGACTC GCTGGTTGGGCA GCAGGATGGGCA CAG CAG DGTGGLHGW 291 CAGAGTGATGGC 585 CAGAGTGACGGA  918 ACCGGGGGGTTG ACAGGAGGACTC CATGGTTGGGCA CACGGATGGGCA CAG CAG DGTGGLQGW 292 CAGAGTGATGGC 586 CAGAGTGACGGA  919 ACCGGGGGTTTG ACAGGAGGACTC CAGGGTTGGGCA CAAGGATGGGCA CAG CAG DGTGGLRGW 154 CAGAGTGATGGC 587 CAGAGTGACGGA  920 ACCGGGGGTTTG ACAGGAGGACTC CGTGGTTGGGCA AGAGGATGGGCA CAG CAG DGTGGLSGW 293 CAGAGTGATGGC 588 CAGAGTGACGGA  921 ACCGGTGGGTTG ACAGGAGGACTC TCGGGTTGGGCA AGCGGATGGGCA CAG CAG DGTGGLTGW 294 CAGAGTGATGGC 589 CAGAGTGACGGA  922 ACCGGGGGGTTG ACAGGAGGACTC ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTGGTKGW 107 CAGAGTGATGGC 590 CAGAGTGACGGA  923 ACCGGTGGGACT ACAGGAGGAACA AAGGGTTGGGCA AAAGGATGGGCA CAG CAG DGTGGTSGW 295 CAGAGTGATGGC 591 CAGAGTGACGGA  924 ACCGGGGGGACG ACAGGAGGAACA AGTGGTTGGGCA AGCGGATGGGCA CAG CAG DGTGGVHGW 296 CAGAGTGATGGC 592 CAGAGTGACGGA  925 ACCGGTGGGGTG ACAGGAGGAGTC CATGGTTGGGCA CACGGATGGGCA CAG CAG DGTGGVMGW 297 CAGAGTGATGGC 593 CAGAGTGACGGA  926 ACCGGTGGTGTT ACAGGAGGAGTC ATGGGGTGGGCA ATGGGATGGGCA CAG CAG DGTGGVSGW 298 CAGAGTGATGGC 594 CAGAGTGACGGA  927 ACCGGGGGGGTG ACAGGAGGAGTC TCTGGTTGGGCAC AGCGGATGGGCA AG CAG DGTGGVTGW 299 CAGAGTGATGGC 595 CAGAGTGACGGA  928 ACCGGTGGTGTG ACAGGAGGAGTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGGVYGW 300 CAGAGTGATGGC 596 CAGAGTGACGGA  929 ACCGGTGGTGTG ACAGGAGGAGTC TATGGGTGGGCA TACGGATGGGCA CAG CAG DGTGNLQGW 301 CAGAGTGATGGC 597 CAGAGTGACGGA  930 ACCGGTAATTTGC ACAGGAAACCTC AGGGTTGGGCAC CAAGGATGGGCA AG CAG DGTGNLRGW 133 CAGAGTGATGGC 598 CAGAGTGACGGA  931 ACCGGGAATCTT ACAGGAAACCTC AGGGGGTGGGCA AGAGGATGGGCA CAG CAG DGTGNLSGW 302 CAGAGTGATGGC 599 CAGAGTGACGGA  932 ACCGGGAATTTG ACAGGAAACCTC AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGNTHGW  72 CAGAGTGATGGC 600 CAGAGTGACGGA  933 ACCGGGAATACT ACAGGAAACACA CATGGGTGGGCA CACGGATGGGCA CAG CAG DGTGNTRGW  94 CAGAGTGATGGC 601 CAGAGTGACGGA  934 ACCGGGAATACT ACAGGAAACACA CGGGGGTGGGCA AGAGGATGGGCA CAG CAG DGTGNTSGW 137 CAGAGTGATGGC 602 CAGAGTGACGGA  935 ACCGGTAATACT ACAGGAAACACA AGTGGTTGGGCA AGCGGATGGGCA CAG CAG DGTGNVSGW 303 CAGAGTGATGGC 603 CAGAGTGACGGA  936 ACCGGGAATGTG ACAGGAAACGTC TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGNVTGW  69 CAGAGTGATGGC 604 CAGAGTGACGGA  937 ACCGGTAATGTG ACAGGAAACGTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGQLVGW 304 CAGAGTGATGGC 605 CAGAGTGACGGA  938 ACCGGGCAGCTT ACAGGACAACTC GTGGGTTGGGCA GTCGGATGGGCA CAG CAG DGTGQTIGW 305 CAGAGTGATGGC 606 CAGAGTGACGGA  939 ACCGGTCAGACG ACAGGACAAACA ATTGGTTGGGCA ATCGGATGGGCA CAG CAG DGTGQVTGW  68 CAGAGTGATGGC 607 CAGAGTGACGGA  940 ACCGGGCAGGTG ACAGGACAAGTC ACTGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGRLTGW 159 CAGAGTGATGGC 608 CAGAGTGACGGA  941 ACCGGTCGGTTG ACAGGAAGACTC ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTGRTVGW 117 CAGAGTGATGGC 609 CAGAGTGACGGA  942 ACCGGTCGGACT ACAGGAAGAACA GTTGGGTGGGCA GTCGGATGGGCA CAG CAG DGTGSGMMT 306 CAGAGTGATGGC 610 CAGAGTGACGGA  943 ACCGGTTCGGGT ACAGGAAGCGGA ATGATGACGGCA ATGATGACAGCA CAG CAG DGTGSISGW 307 CAGAGTGATGGC 611 CAGAGTGACGGA  944 ACCGGGTCGATT ACAGGAAGCATC AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGSLAGW 308 CAGAGTGATGGC 612 CAGAGTGACGGA  945 ACCGGTTCTTTGG ACAGGAAGCCTC CGGGGTGGGCAC GCAGGATGGGCA AG CAG DGTGSLNGW 309 CAGAGTGATGGC 613 CAGAGTGACGGA  946 ACCGGGTCTTTGA ACAGGAAGCCTC ATGGGTGGGCAC AACGGATGGGCA AG CAG DGTGSLQGW 310 CAGAGTGATGGC 614 CAGAGTGACGGA  947 ACCGGGTCGCTG ACAGGAAGCCTC CAGGGTTGGGCA CAAGGATGGGCA CAG CAG DGTGSLSGW 311 CAGAGTGATGGC 615 CAGAGTGACGGA  948 ACCGGGAGTCTG ACAGGAAGCCTC TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGSLVGW 312 CAGAGTGATGGC 616 CAGAGTGACGGA  949 ACCGGGTCGTTG ACAGGAAGCCTC GTGGGTTGGGCA GTCGGATGGGCA CAG CAG DGTGSTHGW 119 CAGAGTGATGGC 617 CAGAGTGACGGA  950 ACCGGGAGTACG ACAGGAAGCACA CATGGGTGGGCA CACGGATGGGCA CAG CAG DGTGSTKGW 313 CAGAGTGATGGC 618 CAGAGTGACGGA  951 ACCGGGAGTACT ACAGGAAGCACA AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGTGSTMGW 314 CAGAGTGATGGC 619 CAGAGTGACGGA  952 ACCGGTTCTACTA ACAGGAAGCACA TGGGTTGGGCAC ATGGGATGGGCA AG CAG DGTGSTQGW 315 CAGAGTGATGGC 620 CAGAGTGACGGA  953 ACCGGTAGTACG ACAGGAAGCACA CAGGGTTGGGCA CAAGGATGGGCA CAG CAG DGTGSTSGW 316 CAGAGTGATGGC 621 CAGAGTGACGGA  954 ACCGGGAGTACT ACAGGAAGCACA TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTGSTTGW 134 CAGAGTGATGGC 622 CAGAGTGACGGA  955 ACCGGGAGTACG ACAGGAAGCACA ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGSVMGW 317 CAGAGTGATGGC 623 CAGAGTGACGGA  956 ACCGGTTCGGTTA ACAGGAAGCGTC TGGGGTGGGCAC ATGGGATGGGCA AG CAG DGTGSVTGW 318 CAGAGTGATGGC 624 CAGAGTGACGGA  957 ACCGGGTCTGTG ACAGGAAGCGTC ACTGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGTLAGW 319 CAGAGTGATGGC 625 CAGAGTGACGGA  958 ACCGGGACGCTT ACAGGAACACTC GCGGGGTGGGCA GCAGGATGGGCA CAG CAG DGTGTLHGW 320 CAGAGTGATGGC 626 CAGAGTGACGGA  959 ACCGGTACTTTGC ACAGGAACACTC ATGGTTGGGCAC CACGGATGGGCA AG CAG DGTGTLKGW 321 CAGAGTGATGGC 627 CAGAGTGACGGA  960 ACCGGTACTCTTA ACAGGAACACTC AGGGTTGGGCAC AAAGGATGGGCA AG CAG DGTGTLSGW 322 CAGAGTGATGGC 628 CAGAGTGACGGA  961 ACCGGGACTCTG ACAGGAACACTC TCGGGTTGGGCA AGCGGATGGGCA CAG CAG DGTGTTLGW 323 CAGAGTGATGGC 629 CAGAGTGACGGA  962 ACCGGGACTACG ACAGGAACAACA CTGGGGTGGGCA CTCGGATGGGCA CAG CAG DGTGTTMGW 324 CAGAGTGATGGC 630 CAGAGTGACGGA  963 ACCGGGACTACT ACAGGAACAACA ATGGGTTGGGCA ATGGGATGGGCA CAG CAG DGTGTTTGW 130 CAGAGTGATGGC 631 CAGAGTGACGGA  964 ACCGGGACTACT ACAGGAACAACA ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTGTTVGW  74 CAGAGTGATGGC 632 CAGAGTGACGGA  965 ACCGGTACTACG ACAGGAACAACA GTGGGGTGGGCA GTCGGATGGGCA CAG CAG DGTGTTYGW 325 CAGAGTGATGGC 633 CAGAGTGACGGA  966 ACCGGGACGACG ACAGGAACAACA TATGGTTGGGCA TACGGATGGGCA CAG CAG DGTGTVHGW 326 CAGAGTGATGGC 634 CAGAGTGACGGA  967 ACCGGTACGGTT ACAGGAACAGTC CATGGTTGGGCA CACGGATGGGCA CAG CAG DGTGTVQGW 327 CAGAGTGATGGC 635 CAGAGTGACGGA  968 ACCGGGACTGTG ACAGGAACAGTC CAGGGGTGGGCA CAAGGATGGGCA CAG CAG DGTGTVSGW 328 CAGAGTGATGGC 636 CAGAGTGACGGA  969 ACCGGTACTGTTT ACAGGAACAGTC CTGGTTGGGCAC AGCGGATGGGCA AG CAG DGTGTVTGW 329 CAGAGTGATGGC 637 CAGAGTGACGGA  970 ACCGGTACTGTTA ACAGGAACAGTC CTGGGTGGGCAC ACAGGATGGGCA AG CAG DGTHARLSS 330 CAGAGTGATGGC 638 CAGAGTGACGGA  971 ACCCATGCGAGG ACACACGCAAGA TTGTCTTCGGCAC CTCAGCAGCGCA AG CAG DGTHAYMAS 153 CAGAGTGATGGC 639 CAGAGTGACGGA  972 ACCCATGCTTATA ACACACGCATAC TGGCGTCTGCAC ATGGCAAGCGCA AG CAG DGTHFAPPR 112 CAGAGTGATGGC 640 CAGAGTGACGGA  973 ACCCATTTTGCGC ACACACTTCGCA CGCCGCGTGCAC CCACCAAGAGCA AG CAG DGTHIHLSS 162 CAGAGTGATGGC 641 CAGAGTGACGGA  974 ACCCATATTCATC ACACACATCCAC TGAGTAGTGCAC CTCAGCAGCGCA AG CAG DGTHIRALS 331 CAGAGTGATGGC 642 CAGAGTGACGGA  975 ACCCATATTAGG ACACACATCAGA GCTCTGAGTGCA GCACTCAGCGCA CAG CAG DGTHIRLAS 332 CAGAGTGATGGC 643 CAGAGTGACGGA  976 ACCCATATTCGTT ACACACATCAGA TGGCGAGTGCAC CTCGCAAGCGCA AG CAG DGTHLQPFR 333 CAGAGTGATGGC 644 CAGAGTGACGGA  977 ACCCATCTGCAG ACACACCTCCAA CCGTTTAGGGCA CCATTCAGAGCA CAG CAG DGTHSFYDA 334 CAGAGTGATGGC 645 CAGAGTGACGGA  978 ACCCATAGTTTTT ACACACAGCTTCT ATGATGCGGCAC ACGACGCAGCAC AG AG DGTHSTTGW 145 CAGAGTGATGGC 646 CAGAGTGACGGA  979 ACCCATTCTACTA ACACACAGCACA CGGGTTGGGCAC ACAGGATGGGCA AG CAG DGTHTRTGW  90 CAGAGTGATGGC 647 CAGAGTGACGGA  980 ACCCATACGCGG ACACACACAAGA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTHVRALS 335 CAGAGTGATGGC 648 CAGAGTGACGGA  981 ACCCATGTTAGG ACACACGTCAGA GCGTTGTCGGCA GCACTCAGCGCA CAG CAG DGTHVYMAS 336 CAGAGTGATGGC 649 CAGAGTGACGGA  982 ACCCATGTTTATA ACACACGTCTAC TGGCTAGTGCAC ATGGCAAGCGCA AG CAG DGTHVYMSS 337 CAGAGTGATGGC 650 CAGAGTGACGGA  983 ACCCATGTGTATA ACACACGTCTAC TGTCTAGTGCACA ATGAGCAGCGCA G CAG DGTIALPFK 338 CAGAGTGATGGC 651 CAGAGTGACGGA  984 ACCATTGCGCTTC ACAATCGCACTC CGTTTAAGGCAC CCATTCAAAGCA AG CAG DGTIALPFR 339 CAGAGTGATGGC 652 CAGAGTGACGGA  985 ACCATTGCTTTGC ACAATCGCACTC CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTIATRYV 340 CAGAGTGATGGC 653 CAGAGTGACGGA  986 ACCATTGCGACG ACAATCGCAACA CGGTATGTGGCA AGATACGTCGCA CAG CAG DGTIERPFR  87 CAGAGTGATGGC 654 CAGAGTGACGGA  987 ACCATTGAGCGG ACAATCGAAAGA CCTTTTCGTGCAC CCATTCAGAGCA AG CAG DGTIGYAYV 341 CAGAGTGATGGC 655 CAGAGTGACGGA  988 ACCATTGGTTATG ACAATCGGATAC CGTATGTTGCACA GCATACGTCGCA G CAG DGTIQAPFK 342 CAGAGTGATGGC 656 CAGAGTGACGGA  989 ACCATTCAGGCTC ACAATCCAAGCA CGTTTAAGGCAC CCATTCAAAGCA AG CAG DGTIRLPFK 343 CAGAGTGATGGC 657 CAGAGTGACGGA  990 ACCATTCGTCTTC ACAATCAGACTC CTTTTAAGGCACA CCATTCAAAGCA G CAG DGTISKEVG 344 CAGAGTGATGGC 658 CAGAGTGACGGA  991 ACCATTTCTAAGG ACAATCAGCAAA AGGTGGGGGCAC GAAGTCGGAGCA AG CAG DGTISQPFK 105 CAGAGTGATGGC 659 CAGAGTGACGGA  992 ACCATTTCGCAGC ACAATCAGCCAA CTTTTAAGGCACA CCATTCAAAGCA G CAG DGTKIQLSS 146 CAGAGTGATGGC 660 CAGAGTGACGGA  993 ACCAAGATTCAG ACAAAAATCCAA CTGTCTAGTGCAC CTCAGCAGCGCA AG CAG DGTKIRLSS 111 CAGAGTGATGGC 661 CAGAGTGACGGA  994 ACCAAGATTCGG ACAAAAATCAGA TTGTCGTCTGCAC CTCAGCAGCGCA AG CAG DGTKLMLSS 157 CAGAGTGATGGC 662 CAGAGTGACGGA  995 ACCAAGCTGATG ACAAAACTCATG TTGAGTAGTGCA CTCAGCAGCGCA CAG CAG DGTKLRLSS 118 CAGAGTGATGGC 663 CAGAGTGACGGA  996 ACCAAGTTGAGG ACAAAACTCAGA CTTAGTTCTGCAC CTCAGCAGCGCA AG CAG DGTKMVLQL 142 CAGAGTGATGGC 664 CAGAGTGACGGA  997 ACCAAGATGGTG ACAAAAATGGTC TTGCAGCTGGCA CTCCAACTCGCAC CAG AG DGTKSLVQL 345 CAGAGTGATGGC 665 CAGAGTGACGGA  998 ACCAAGAGTCTT ACAAAAAGCCTC GTGCAGCTTGCA GTCCAACTCGCA CAG CAG DGTKVLVQL 122 CAGAGTGATGGC 666 CAGAGTGACGGA  999 ACCAAGGTGCTG ACAAAAGTCCTC GTGCAGTTGGCA GTCCAACTCGCA CAG CAG DGTLAAPFK 120 CAGAGTGATGGC 667 CAGAGTGACGGA 1000 ACCTTGGCTGCTC ACACTCGCAGCA CTTTTAAGGCACA CCATTCAAAGCA G CAG DGTLAVNFK 346 CAGAGTGATGGG 668 CAGAGTGACGGA 1001 ACTTTGGCGGTG ACACTCGCAGTC AATTTTAAGGCA AACTTCAAAGCA CAG CAG DGTLAVPFK  71 CAGAGTGATGGG 669 CAGAGTGACGGA 1002 (PHP.eB) ACTTTGGCGGTGC ACACTCGCAGTC CTTTTAAGGCACA CCATTCAAAGCA G CAG DGTLAYPFK 347 CAGAGTGATGGC 670 CAGAGTGACGGA 1003 ACCCTTGCGTATC ACACTCGCATAC CTTTTAAGGCACA CCATTCAAAGCA G CAG DGTLERPFR 156 CAGAGTGATGGC 671 CAGAGTGACGGA 1004 ACCCTGGAGAGG ACACTCGAAAGA CCGTTTCGGGCAC CCATTCAGAGCA AG CAG DGTLEVHFK 348 CAGAGTGATGGG 672 CAGAGTGACGGA 1005 ACTTTGGAGGTG ACACTCGAAGTC CATTTTAAGGCAC CACTTCAAAGCA AG CAG DGTLLRLSS 121 CAGAGTGATGGC 673 CAGAGTGACGGA 1006 ACCTTGCTGAGG ACACTCCTCAGA CTGAGTAGTGCA CTCAGCAGCGCA CAG CAG DGTLNNPFR 109 CAGAGTGATGGC 674 CAGAGTGACGGA 1007 ACCTTGAATAATC ACACTCAACAAC CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTLQQPFR  89 CAGAGTGATGGC 675 CAGAGTGACGGA 1008 ACCTTGCAGCAG ACACTCCAACAA CCGTTTCGGGCAC CCATTCAGAGCA AG CAG DGTLSQPFR  65 CAGAGTGATGGC 676 CAGAGTGACGGA 1009 ACCCTGTCTCAGC ACACTCAGCCAA CTTTTAGGGCACA CCATTCAGAGCA G CAG DGTLSRTLW 349 CAGAGTGATGGC 677 CAGAGTGACGGA 1010 ACCTTGTCGCGTA ACACTCAGCAGA CGCTTTGGGCAC ACACTCTGGGCA AG CAG DGTLSSPFR 350 CAGAGTGATGGC 678 CAGAGTGACGGA 1011 ACCCTGTCTAGTC ACACTCAGCAGC CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTLTVPFR 351 CAGAGTGATGGC 679 CAGAGTGACGGA 1012 ACCTTGACGGTTC ACACTCACAGTC CTTTTCGGGCACA CCATTCAGAGCA G CAG DGTLVAPFR 352 CAGAGTGATGGC 680 CAGAGTGACGGA 1013 ACCCTTGTTGCGC ACACTCGTCGCA CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTMDKPFR  70 CAGAGTGATGGC 681 CAGAGTGACGGA 1014 ACGATGGATAAG ACAATGGACAAA CCTTTTAGGGCAC CCATTCAGAGCA AG CAG DGTMDRPFK 102 CAGAGTGATGGC 682 CAGAGTGACGGA 1015 ACCATGGATAGG ACAATGGACAGA CCGTTTAAGGCA CCATTCAAAGCA CAG CAG DGTMLRLSS 148 CAGAGTGATGGC 683 CAGAGTGACGGA 1016 ACCATGTTGCGTC ACAATGCTCAGA TTAGTTCGGCACA CTCAGCAGCGCA G CAG DGTMQLTGW 353 CAGAGTGATGGC 684 CAGAGTGACGGA 1017 ACCATGCAGCTT ACAATGCAACTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTNGLKGW  76 CAGAGTGATGGC 685 CAGAGTGACGGA 1018 ACCAATGGTCTG ACAAACGGACTC AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGTNSISGW 354 CAGAGTGATGGC 686 CAGAGTGACGGA 1019 ACCAATAGTATT ACAAACAGCATC AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGTNSLSGW 355 CAGAGTGATGGC 687 CAGAGTGACGGA 1020 ACCAATTCTCTGT ACAAACAGCCTC CGGGTTGGGCAC AGCGGATGGGCA AG CAG DGTNSTTGW 143 CAGAGTGATGGC 688 CAGAGTGACGGA 1021 ACCAATTCTACG ACAAACAGCACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTNSVTGW 356 CAGAGTGATGGC 689 CAGAGTGACGGA 1022 ACCAATAGTGTT ACAAACAGCGTC ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTNTINGW 124 CAGAGTGATGGC 690 CAGAGTGACGGA 1023 ACCAATACTATTA ACAAACACAATC ATGGGTGGGCAC AACGGATGGGCA AG CAG DGTNTLGGW 357 CAGAGTGATGGC 691 CAGAGTGACGGA 1024 ACCAATACGTTG ACAAACACACTC GGGGGGTGGGCA GGAGGATGGGCA CAG CAG DGTNTTHGW 113 CAGAGTGATGGC 692 CAGAGTGACGGA 1025 ACCAATACTACTC ACAAACACAACA ATGGGTGGGCAC CACGGATGGGCA AG CAG DGTNYRLSS 358 CAGAGTGATGGC 693 CAGAGTGACGGA 1026 ACCAATTATAGG ACAAACTACAGA CTGTCGAGTGCA CTCAGCAGCGCA CAG CAG DGTQALSGW 359 CAGAGTGATGGC 694 CAGAGTGACGGA 1027 ACCCAGGCGCTG ACACAAGCACTC TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTQFRLSS 129 CAGAGTGATGGC 695 CAGAGTGACGGA 1028 ACCCAGTTTAGGT ACACAATTCAGA TGTCTTCGGCACA CTCAGCAGCGCA G CAG DGTQFSPPR 108 CAGAGTGATGGC 696 CAGAGTGACGGA 1029 ACCCAGTTTAGTC ACACAATTCAGC CTCCGCGTGCAC CCACCAAGAGCA AG CAG DGTQGLKGW 158 CAGAGTGATGGC 697 CAGAGTGACGGA 1030 ACCCAGGGGCTG ACACAAGGACTC AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGTQTTSGW 360 CAGAGTGATGGC 698 CAGAGTGACGGA 1031 ACCCAGACTACG ACACAAACAACA AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGTRALTGW 361 CAGAGTGATGGC 699 CAGAGTGACGGA 1032 ACCAGGGCTCTT ACAAGAGCACTC ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTRFSLSS 362 CAGAGTGATGGC 700 CAGAGTGACGGA 1033 ACCCGGTTTTCGC ACAAGATTCAGC TTTCGAGTGCACA CTCAGCAGCGCA G CAG DGTRGLSGW 363 CAGAGTGATGGC 701 CAGAGTGACGGA 1034 ACCAGGGGGTTG ACAAGAGGACTC TCGGGGTGGGCA AGCGGATGGGCA CAG CAG DGTRIGLSS 364 CAGAGTGATGGC 702 CAGAGTGACGGA 1035 ACCAGGATTGGG ACAAGAATCGGA CTGAGTAGTGCA CTCAGCAGCGCA CAG CAG DGTRLHLAS 365 CAGAGTGATGGC 703 CAGAGTGACGGA 1036 ACCAGGCTTCATC ACAAGACTCCAC TGGCGAGTGCAC CTCGCAAGCGCA AG CAG DGTRLHLSS 366 CAGAGTGATGGC 704 CAGAGTGACGGA 1037 ACCAGGCTTCATC ACAAGACTCCAC TGTCGTCGGCAC CTCAGCAGCGCA AG CAG DGTRLLLSS 367 CAGAGTGATGGC 705 CAGAGTGACGGA 1038 ACCCGTTTGCTGC ACAAGACTCCTC TGTCGAGTGCAC CTCAGCAGCGCA AG CAG DGTRLMLSS 368 CAGAGTGATGGC 706 CAGAGTGACGGA 1039 ACCCGTTTGATGC ACAAGACTCATG TTTCTAGTGCACA CTCAGCAGCGCA G CAG DGTRLNLSS 369 CAGAGTGATGGC 707 CAGAGTGACGGA 1040 ACCCGTTTGAATC ACAAGACTCAAC TTAGTTCGGCACA CTCAGCAGCGCA G CAG DGTRMVVQL 370 CAGAGTGATGGC 708 CAGAGTGACGGA 1041 ACCCGGATGGTT ACAAGAATGGTC GTTCAGCTTGCAC GTCCAACTCGCA AG CAG DGTRNMYEG 135 CAGAGTGATGGC 709 CAGAGTGACGGA 1042 ACCCGTAATATGT ACAAGAAACATG ATGAGGGGGCAC TACGAAGGAGCA AG CAG DGTRSITGW 371 CAGAGTGATGGC 710 CAGAGTGACGGA 1043 ACCAGGAGTATT ACAAGAAGCATC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTRSLHGW 372 CAGAGTGATGGC 711 CAGAGTGACGGA 1044 ACCAGGAGTTTG ACAAGAAGCCTC CATGGGTGGGCA CACGGATGGGCA CAG CAG DGTRSTTGW 373 CAGAGTGATGGC 712 CAGAGTGACGGA 1045 ACCCGGAGTACT ACAAGAAGCACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTRTTTGW 106 CAGAGTGATGGC 713 CAGAGTGACGGA 1046 ACCCGTACTACG ACAAGAACAACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTRTVTGW 374 CAGAGTGATGGC 714 CAGAGTGACGGA 1047 ACCCGGACGGTG ACAAGAACAGTC ACTGGTTGGGCA ACAGGATGGGCA CAG CAG DGTRTVVQL 375 CAGAGTGATGGC 715 CAGAGTGACGGA 1048 ACCCGTACTGTG ACAAGAACAGTC GTGCAGTTGGCA GTCCAACTCGCA CAG CAG DGTRVHLSS 376 CAGAGTGATGGC 716 CAGAGTGACGGA 1049 ACCCGGGTGCAT ACAAGAGTCCAC CTTTCTAGTGCAC CTCAGCAGCGCA AG CAG DGTSFPYAR  86 CAGAGTGATGGC 717 CAGAGTGACGGA 1050 ACCTCGTTTCCGT ACAAGCTTCCCAT ATGCTCGGGCAC ACGCAAGAGCAC AG AG DGTSFTPPK  81 CAGAGTGATGGC 718 CAGAGTGACGGA 1051 ACCTCGTTTACGC ACAAGCTTCACA CGCCTAAGGCAC CCACCAAAAGCA AG CAG DGTSFTPPR  88 CAGAGTGATGGC 719 CAGAGTGACGGA 1052 ACCTCGTTTACTC ACAAGCTTCACA CGCCGCGGGCAC CCACCAAGAGCA AG CAG DGTSGLHGW 377 CAGAGTGATGGC 720 CAGAGTGACGGA 1053 ACCTCTGGGTTGC ACAAGCGGACTC ATGGGTGGGCAC CACGGATGGGCA AG CAG DGTSGLKGW 101 CAGAGTGATGGC 721 CAGAGTGACGGA 1054 ACCAGTGGGCTT ACAAGCGGACTC AAGGGGTGGGCA AAAGGATGGGCA CAG CAG DGTSIHLSS 378 CAGAGTGATGGC 722 CAGAGTGACGGA 1055 ACCTCGATTCATT ACAAGCATCCAC TGAGTAGTGCAC CTCAGCAGCGCA AG CAG DGTSIMLSS 379 CAGAGTGATGGC 723 CAGAGTGACGGA 1056 ACCTCGATTATGT ACAAGCATCATG TGAGTTCTGCACA CTCAGCAGCGCA G CAG DGTSLRLSS 166 CAGAGTGATGGC 724 CAGAGTGACGGA 1057 ACCTCTTTGCGGC ACAAGCCTCAGA TTTCTTCTGCACA CTCAGCAGCGCA G CAG DGTSNYGAR 380 CAGAGTGATGGC 725 CAGAGTGACGGA 1058 ACCTCTAATTATG ACAAGCAACTAC GGGCGCGGGCAC GGAGCAAGAGCA AG CAG DGTSSYYDA 381 CAGAGTGATGGC 726 CAGAGTGACGGA 1059 ACCAGTTCGTATT ACAAGCAGCTAC ATGATGCGGCAC TACGACGCAGCA AG CAG DGTSSYYDS  59 CAGAGTGATGGC 727 CAGAGTGACGGA 1060 ACCTCGAGTTATT ACAAGCAGCTAC ATGATTCTGCACA TACGACAGCGCA G CAG DGTSTISGW 382 CAGAGTGATGGC 728 CAGAGTGACGGA 1061 ACCTCTACGATTT ACAAGCACAATC CTGGTTGGGCAC AGCGGATGGGCA AG CAG DGTSTITGW 383 CAGAGTGATGGC 729 CAGAGTGACGGA 1062 ACCAGTACTATTA ACAAGCACAATC CGGGTTGGGCAC ACAGGATGGGCA AG CAG DGTSTLHGW 384 CAGAGTGATGGC 730 CAGAGTGACGGA 1063 ACCTCGACGTTGC ACAAGCACACTC ATGGGTGGGCAC CACGGATGGGCA AG CAG DGTSTLRGW 385 CAGAGTGATGGC 731 CAGAGTGACGGA 1064 ACCTCTACTCTGC ACAAGCACACTC GTGGGTGGGCAC AGAGGATGGGCA AG CAG DGTSTLSGW 386 CAGAGTGATGGC 732 CAGAGTGACGGA 1065 ACCTCGACGCTGT ACAAGCACACTC CGGGGTGGGCAC AGCGGATGGGCA AG CAG DGTSYVPPK  97 CAGAGTGATGGC 733 CAGAGTGACGGA 1066 ACCTCTTATGTGC ACAAGCTACGTC CGCCGAAGGCAC CCACCAAAAGCA AG CAG DGTSYVPPR  78 CAGAGTGATGGC 734 CAGAGTGACGGA 1067 ACCAGTTATGTGC ACAAGCTACGTC CGCCTCGGGCAC CCACCAAGAGCA AG CAG DGTTATYYK 387 CAGAGTGATGGC 735 CAGAGTGACGGA 1068 ACCACGGCGACT ACAACAGCAACA TATTATAAGGCA TACTACAAAGCA CAG CAG DGTTFTPPR  79 CAGAGTGATGGC 736 CAGAGTGACGGA 1069 ACCACTTTTACTC ACAACATTCACA CTCCTCGGGCAC CCACCAAGAGCA AG CAG DGTTLAPFR 388 CAGAGTGATGGC 737 CAGAGTGACGGA 1070 ACCACTCTGGCTC ACAACACTCGCA CTTTTAGGGCACA CCATTCAGAGCA G CAG DGTTLVPPR 116 CAGAGTGATGGC 738 CAGAGTGACGGA 1071 ACCACTTTGGTTC ACAACACTCGTC CGCCGCGTGCAC CCACCAAGAGCA AG CAG DGTTSKTLW 389 CAGAGTGATGGC 739 CAGAGTGACGGA 1072 ACCACGAGTAAG ACAACAAGCAAA ACGCTTTGGGCA ACACTCTGGGCA CAG CAG DGTTSRTLW 390 CAGAGTGATGGC 740 CAGAGTGACGGA 1073 ACCACTTCTAGG ACAACAAGCAGA ACTTTGTGGGCAC ACACTCTGGGCA AG CAG DGTTTRSLY 391 CAGAGTGATGGC 741 CAGAGTGACGGA 1074 ACCACGACTCGT ACAACAACAAGA AGTTTGTATGCAC AGCCTCTACGCA AG CAG DGTTTTTGW 392 CAGAGTGATGGC 742 CAGAGTGACGGA 1075 ACCACTACGACT ACAACAACAACA ACGGGTTGGGCA ACAGGATGGGCA CAG CAG DGTTTYGAR  77 CAGAGTGATGGC 743 CAGAGTGACGGA 1076 ACCACTACGTAT ACAACAACATAC GGGGCTCGTGCA GGAGCAAGAGCA CAG CAG DGTTWTPPR 139 CAGAGTGATGGC 744 CAGAGTGACGGA 1077 ACCACTTGGACG ACAACATGGACA CCGCCGCGTGCA CCACCAAGAGCA CAG CAG DGTTYMLSS 393 CAGAGTGATGGC 745 CAGAGTGACGGA 1078 ACCACGTATATG ACAACATACATG CTTAGTAGTGCAC CTCAGCAGCGCA AG CAG DGTTYVPPR  75 CAGAGTGATGGC 746 CAGAGTGACGGA 1079 ACCACGTATGTTC ACAACATACGTC CTCCGCGGGCAC CCACCAAGAGCA AG CAG DGTVANPFR 394 CAGAGTGATGGC 747 CAGAGTGACGGA 1080 ACCGTGGCGAAT ACAGTCGCAAAC CCTTTTCGGGCAC CCATTCAGAGCA AG CAG DGTVDRPFK 395 CAGAGTGATGGC 748 CAGAGTGACGGA 1081 ACCGTGGATCGG ACAGTCGACAGA CCTTTTAAGGCAC CCATTCAAAGCA AG CAG DGTVIHLSS  73 CAGAGTGATGGC 749 CAGAGTGACGGA 1082 ACCGTTATTCATC ACAGTCATCCAC TGAGTAGTGCAC CTCAGCAGCGCA AG CAG DGTVILLSS 396 CAGAGTGATGGC 750 CAGAGTGACGGA 1083 ACCGTTATTCTGT ACAGTCATCCTCC TGTCGAGTGCAC TCAGCAGCGCAC AG AG DGTVIMLSS 397 CAGAGTGATGGC 751 CAGAGTGACGGA 1084 ACCGTGATTATGC ACAGTCATCATG TGTCGAGTGCAC CTCAGCAGCGCA AG CAG DGTVLHLSS 398 CAGAGTGATGGC 752 CAGAGTGACGGA 1085 ACCGTGCTTCATT ACAGTCCTCCACC TGTCGTCTGCACA TCAGCAGCGCAC G AG DGTVLMLSS 399 CAGAGTGATGGC 753 CAGAGTGACGGA 1086 ACCGTTTTGATGC ACAGTCCTCATGC TGAGTAGTGCAC TCAGCAGCGCAC AG AG DGTVLVPFR 150 CAGAGTGATGGC 754 CAGAGTGACGGA 1087 ACCGTGTTGGTGC ACAGTCCTCGTCC CGTTTAGGGCAC CATTCAGAGCAC AG AG DGTVPYLAS 400 CAGAGTGATGGC 755 CAGAGTGACGGA 1088 ACCGTTCCGTATC ACAGTCCCATAC TTGCTTCTGCACA CTCGCAAGCGCA G CAG DGTVPYLSS 401 CAGAGTGATGGC 756 CAGAGTGACGGA 1089 ACCGTGCCGTATT ACAGTCCCATAC TGTCTTCGGCACA CTCAGCAGCGCA G CAG DGTVRVPFR 164 CAGAGTGATGGC 757 CAGAGTGACGGA 1090 ACCGTTCGTGTGC ACAGTCAGAGTC CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTVSMPFK 402 CAGAGTGATGGC 758 CAGAGTGACGGA 1091 ACCGTGTCGATG ACAGTCAGCATG CCGTTTAAGGCA CCATTCAAAGCA CAG CAG DGTVSNPFR 403 CAGAGTGATGGC 759 CAGAGTGACGGA 1092 ACCGTGTCTAATC ACAGTCAGCAAC CGTTTAGGGCAC CCATTCAGAGCA AG CAG DGTVSTRWV 404 CAGAGTGATGGC 760 CAGAGTGACGGA 1093 ACCGTTTCTACGC ACAGTCAGCACA GTTGGGTGGCAC AGATGGGTCGCA AG CAG DGTVTTTGW 405 CAGAGTGATGGC 761 CAGAGTGACGGA 1094 ACCGTGACGACG ACAGTCACAACA ACTGGGTGGGCA ACAGGATGGGCA CAG CAG DGTVTVTGW 406 CAGAGTGATGGC 762 CAGAGTGACGGA 1095 ACCGTGACGGTT ACAGTCACAGTC ACGGGGTGGGCA ACAGGATGGGCA CAG CAG DGTVWVPPR 407 CAGAGTGATGGC 763 CAGAGTGACGGA 1096 ACCGTTTGGGTGC ACAGTCTGGGTC CTCCTAGGGCAC CCACCAAGAGCA AG CAG DGTVYRLSS 408 CAGAGTGATGGC 764 CAGAGTGACGGA 1097 ACCGTTTATAGGT ACAGTCTACAGA TGTCGAGTGCAC CTCAGCAGCGCA AG CAG DGTYARLSS 409 CAGAGTGATGGC 765 CAGAGTGACGGA 1098 ACCTATGCGCGTT ACATACGCAAGA TGTCTTCTGCACA CTCAGCAGCGCA G CAG DGTYGNKLW 410 CAGAGTGATGGC 766 CAGAGTGACGGA 1099 ACCTATGGTAAT ACATACGGAAAC AAGTTGTGGGCA AAACTCTGGGCA CAG CAG DGTYIHLSS 411 CAGAGTGATGGC 767 CAGAGTGACGGA 1100 ACCTATATTCATC ACATACATCCAC TGTCTTCGGCACA CTCAGCAGCGCA G CAG DGTYSTSGW 412 CAGAGTGATGGC 768 CAGAGTGACGGA 1101 ACCTATTCGACG ACATACAGCACA AGTGGGTGGGCA AGCGGATGGGCA CAG CAG DGVHPGLSS 104 CAGAGTGATGGC 769 CAGAGTGACGGA 1102 GTGCATCCTGGG GTCCACCCAGGA CTTTCGAGTGCAC CTCAGCAGCGCA AG CAG DGVVALLAS 413 CAGAGTGATGGC 770 CAGAGTGACGGA 1103 GTGGTTGCGTTGC GTCGTCGCACTCC TTGCTAGTGCACA TCGCAAGCGCAC G AG DGYVGVGSL 414 CAGAGTGATGGC 771 CAGAGTGACGGA 1104 TATGTGGGTGTTG TACGTCGGAGTC GTAGTTTGGCAC GGAAGCCTCGCA AG CAG

Primer pools were produced by Twist biosciences using solid-phase synthesis and were used to generate a balanced library of 666 nucleotide variants by PCR amplification of CAP C-terminus and Gibson assembly as described in FIG. 27. 666 primers were provided a 1 fmole each, resulting in 0.6 pmole (regular PCR requires ˜25 pmole of primer). Primerless amplification on capsid gBlock template was performed over 10 cycles. Forward and reverse primers were added, followed by an additional 10, 15 or 20 PCR cycles. Constructs were then cloned into AAV9 backbone plasmids by Gibson/RCA (like regular libraries).

NGS analysis of SYN- and GFAP-driven AAV libraries produced with the pooled DNA showed a good correlation between the codon variants of each peptide, suggesting that the DNA sequence itself had little influence on virus production (FIG. 28 and FIG. 29). The pooled synthetic library was injected intravenously to C57BL/6 mice (5e11 VG per mouse, N=9), BALB/C mice (5e11 VG per mouse, N=6) and to rats (5e12 VG per rat, N=6), and after one month in-life RNA was extracted from the brain and spinal cord, and DNA was extracted from liver and heart tissue samples for biodistribution analysis (FIG. 30). Because the Synapsin and GFAP promoters are not fully active in non-CNS tissue, DNA was analyzed instead of RNA in peripheral organs. The initial focus was on the C57BL/6 mouse analysis because this is the mouse strain in which library evolution was performed.

The enrichment score of each capsid was determined by NGS analysis and defined as the ratio of reads per million (RPM) in the target tissue versus RPM in the inoculum. An example of analysis performed on the control capsids is shown in FIG. 31A. As expected from the published data, the PHP.B and PHP.eB (aka, PHP.N) capsids allowed significantly higher RNA expression in neurons compared to the AAV9 parental capsid (8-fold and 25-fold, respectively). There was a very high correlation between the codon variants of each peptide species in each animal (r=0.92, 0.93 and 0.95), confirming the robustness of the NGS assay (FIG. 31B-FIG. 31D).

An example of enrichment analysis is presented in FIG. 32A-FIG. 36. The 333 capsid variants are ranked by average brain enrichment score from all animals, and the individual enrichment values are indicated by a color scale. As indicated by the position of the reference capsids, a group of novel variants showed a higher enrichment score than the PHP.eB benchmark capsid in both neurons (Syn-driven) and astrocytes (GFAP-driven). Interestingly, many variants showed a different enrichment score in neurons vs. astrocytes, as indicated by the medium level of correlation between Syn- and GFAP-driven RNA. This suggests that certain capsids display an enhanced tropism for neurons, and others for astrocytes (FIG. 33).

A group of 38 capsids showed potentially interesting properties based on their tropism for neurons, astrocytes or both (Table 8A and Table 8B) (FIG. 38) and showed a strong consensus peptide sequence similarity, different between neuron- and astrocyte-targeting variants (FIG. 45A-FIG. 45C and FIG. 46A-FIG. 46B).

TABLE 8A TOP 38 candidates from C57BL/6 screen #1 (N =3) SEQ ID SYN GFAP Groups variant peptide NO: ranking ranking A 9p32 DGTAIHLSS  67 15, 16 113, 133 9p35 DGTSSYYDS  59 1, 3 565, 581 B 9p36 DGSSSYYDA  64 10, 11 591, 594 9p37 DGTASYYDS  61 5, 6 553, 560 C 9p26 DGTTTYGAR  77 225, 262 49, 56 D 9p2 AQNGNPGRW  84 156, 160 38, 44 9p13 AQGENPGRW  96 77, 87 7, 13 9p30 AQPEGSARW  60 2, 4 154, 160 E 9p1 AQGSWNPPA  80 348, 361 8, 15 9p14 AQGTWNPPA  82 448, 467 14, 17 F 9p29 AQFPTNYDS  66 14, 19 490, 537 9p31 AQWPTSYDA  62 7, 9 290, 304 G 9p3 AQTTEKPWL  83 53, 72 35, 70 9p15 AQTTDRPFL  85 206, 219 26, 43 H 9p10 DGTRTTTGW 106 161, 220 10, 22 9p18 DGTGGIKGW 131 346, 388 41, 68 9p19 DGTGNTRGW  94 322, 340 45, 54 9p20 DGTHTRTGW  90 380, 427 31, 39 9p23 DGTNGLKGW  76 132, 153 5, 16 9p33 DGTGQVTGW  68 18, 33 172, 213 9p38 DGTGNVTGW  69 20, 31 117, 137 I 9p11 DGTTFTPPR  79 183, 199 11, 19 9p12 DGTTYVPPR  75 146, 154 4, 9 9p24 DGTSFTPPK  81 210, 243 29, 40 9p25 DGTSFTPPR  88 250, 273 28, 37 9p27 DGTTWTPPR 139 567, 570 46, 59 9p28 DGTSYVPPR  78 162, 179 20, 25 J 9p4 DGTADRPFR 155 109, 118 48, 57 9p9 DGTMDRPFK 102 102, 113 23 ,34 9p16 DGTADKPFR  63 8, 12 1, 6 9p17 DGTAERPFR 140 106, 138 42, 50 9p21 DGTIERPFR  87 186, 235 21, 33 9p34 DGTMDKPFR  70 21, 23 107, 112 K 9p5 DGTISQPFK 105 184, 193 12, 18 9p6 DGTLAAPFK 120 110, 112 27, 30 9p7 DGTLQQPFR  89 46, 57 32, 47 9p8 DGTLSQPFR  65 13, 17 2, 3 9p22 DGTLNNPFR 109 30, 41 24, 36 Ref. PHPN DGTLAVPFK  71 22, 24 51, 60 PHPB AQTLAVPFK 168 253, 261 61, 62 wtAAV9 AQ 630, 631 611, 620

TABLE 8B Variant 9mer and encoding sequences SEQ NNK  SEQ NNM SEQ 9mer  ID nucleotide ID nucleotide ID variant peptide NO: sequences NO: sequences NO: 9p1 AQGSWNPPA  80 GCCCAAGGTT 1105 GCACAAGGAAG 1143 CGTGGAATCC CTGGAACCCACC GCCGGCG AGCA 9p2 AQNGNPGRW  84 GCCCAAAATG 1106 GCACAAAACGG 1144 GTAATCCGGG AAACCCAGGAA GCGGTGG GATGG 9p3 AQTIEKPWL  83 GCCCAAACGA 1107 GCACAAACAAC 1145 CTGAGAAGCC AGAAAAACCAT GTGGCTG GGCTC 9p4 DGTADRPFR 155 GATGGCACGG 1108 GACGGAACAGC 1146 CGGATCGTCCT AGACAGACCATT TTTCGG CAGA 9p5 DGTISQPFK 105 GATGGCACCA 1109 GACGGAACAAT 1147 TTTCGCAGCCT CAGCCAACCATT TTTAAG CAAA 9p6 DGTLAAPFK 120 GATGGCACCTT 1110 GACGGAACACTC 1148 GGCTGCTCCTT GCAGCACCATTC TTAAG AAA 9p7 DGTLQQPFR  89 GATGGCACCTT 1111 GACGGAACACTC 1149 GCAGCAGCCG CAACAACCATTC TTTCGG AGA 9p8 DGTLSQPFR  65 GATGGCACCC 1112 GACGGAACACTC 1150 TGTCTCAGCCT AGCCAACCATTC TTTAGG AGA 9p9 DGTMDRPFK 102 GATGGCACCA 1113 GACGGAACAAT 1151 TGGATAGGCC GGACAGACCATT GTTTAAG CAAA 9p10 DGTRTTTGW 106 GATGGCACCC 1114 GACGGAACAAG 1152 GTACTACGAC AACAACAACAG GGGTTGG GATGG 9p11 DGTTFTPPR  79 GATGGCACCA 1115 GACGGAACAAC 1153 CTTTTACTCCT ATTCACACCACC CCTCGG AAGA 9p12 DGTTYVPPR  75 GATGGCACCA 1116 GACGGAACAAC 1154 CGTATGTTCCT ATACGTCCCACC CCGCGG AAGA 9p13 AQGENPGRW  96 GCCCAAGGGG 1117 GCACAAGGAGA 1155 AGAATCCGGG AAACCCAGGAA TAGGTGG GATGG 9p14 AQGTWNPPA  82 GCCCAAGGTA 1118 GCACAAGGAAC 1156 CTTGGAATCCG ATGGAACCCACC CCGGCT AGCA 9p15 AQTTDRPFL  85 GCCCAAACTA 1119 GCACAAACAAC 1157 CTGATAGGCCT AGACAGACCATT TTTTTG CCTC 9p16 DGTADKPFR  63 GATGGCACCG 1120 GACGGAACAGC 1158 CTGATAAGCC AGACAAACCATT GTTTCGG CAGA 9p17 DGTAERPFR 140 GATGGCACCG 1121 GACGGAACAGC 1159 CGGAGAGGCC AGAAAGACCATT TTTTAGG CAGA 9p18 DGTGGIKGW 131 GATGGCACCG 1122 GACGGAACAGG 1160 GTGGTATTAA AGGAATCAAAG GGGGTGG GATGG 9p19 DGTGNTRGW  94 GATGGCACCG 1123 GACGGAACAGG 1161 GGAATACTCG AAACACAAGAG GGGGTGG GATGG 9p20 DGTHTRTGW  90 GATGGCACCC 1124 GACGGAACACA 1162 ATACGCGGAC CACAAGAACAG GGGTTGG GATGG 9p21 DGTIERPFR  87 GATGGCACCA 1125 GACGGAACAAT 1163 TTGAGCGGCCT CGAAAGACCATT TTTCGT CAGA 9p22 DGTLNNPFR 109 GATGGCACCTT 1126 GACGGAACACTC 1164 GAATAATCCG AACAACCCATTC TTTAGG AGA 9p23 DGTNGLKGW  76 GATGGCACCA 1127 GACGGAACAAA 1165 ATGGTCTGAA CGGACTCAAAG GGGGTGG GATGG 9p24 DGTSFTPPK  81 GATGGCACCT 1128 GACGGAACAAG 1166 CGTTTACGCCG CTTCACACCACC CCTAAG AAAA 9p25 DGTSFTPPR  88 GATGGCACCT 1129 GACGGAACAAG 1167 CGTTTACTCCG CTTCACACCACC CCGCGG AAGA 9p26 DGTTTYGAR  77 GATGGCACCA 1130 GACGGAACAAC 1168 CTACGTATGG AACATACGGAG GGCTCGT CAAGA 9p27 DGTTWTPPR 139 GATGGCACCA 1131 GACGGAACAAC 1169 CTTGGACGCC ATGGACACCACC GCCGCGT AAGA 9p28 DGTSYVPPR  78 GATGGCACCA 1132 GACGGAACAAG 1170 GTTATGTTCCT CTACGTCCCACC CCGAGG AAGA 9p29 AQFPTNYDS  66 GCCCAATTTCC 1133 GCACAATTCCCA 1171 TACGAATTATG ACAAACTACGAC ATTCT AGC 9p30 AQPEGSARW  60 GCCCAACCTG 1134 GCACAACCAGA 1172 AGGGTAGTGC AGGAAGCGCAA GAGGTGG GATGG 9p31 AQWPTSYDA  62 GCCCAATGGC 1135 GCACAATGGCCA 1173 CTACGAGTTAT ACAAGCTACGAC GATGCT GCA 9p32 DGTAIHLSS  67 GATGGCACCG 1136 GACGGAACAGC 1174 CGATTCATCTT AATCCACCTCAG TCGTCT CAGC 9p33 DGTGQVTGW  68 GATGGCACCG 1137 GACGGAACAGG 1175 GGCAGGTGAC ACAAGTCACAG TGGGTGG GATGG 9p34 DGTMDKPFR  70 GATGGCACGA 1138 GACGGAACAAT 1176 TGGATAAGCC GGACAAACCATT TTTTAGG CAGA 9p35 DGTSSYYDS  59 GATGGCACCT 1139 GACGGAACAAG 1177 CGAGTTATTAT CAGCTACTACGA GATTCT CAGC 9p36 DGSSSYYDA  64 GATGGCAGTA 1140 GACGGAAGCAG 1178 GTTCTTATTAT CAGCTACTACGA GATGCG CGCA 9p37 DGTASYYDS  61 GATGGCACCG 1141 GACGGAACAGC 1179 CGAGTTATTAT AAGCTACTACGA GATTCT CAGC 9p38 DGTGNVTGW  69 GATGGCACCG 1142 GACGGAACAGG 1180 GTAATGTGAC AAACGTCACAG GGGGTGG GATGG AAV9 AQ AGTGCTCAGG   54 AGTGCCCAAGCA   53 CACAGGCGCA CAGGCGCAGAC GACC C PHPN DGTLAVPFK  71 GATGGGACTTT   56 GACGGAACACTC   55 GGCGGTGCCTT GCAGTCCCATTC TTAAG AAA PHPB AQTLAVPFK 168 GCCCAAACTTT   58 GCACAAACACTC   57 GGCGGTGCCTT GCAGTCCCATTC TTAAG AAA

Example 11. Phylogenetic Grouping

Phylogenetic grouping of peptide sequences showed an evident correlation between sequence homology clusters and capsid phenotypes (FIG. 37). For example, 9-mer variants with the sequence DGTxxxPFK/R (SEQ ID NO: 1181) presented a similar behavior as PHP.eB capsid (high transduction of both neurons and astrocytes), whereas variants harboring the sequence DGTxxxYDS/A (SEQ ID NO: 1182) showed a preference for neuron transduction. By contrast, peptides with the consensus DGTxxxxGW (SEQ ID NO: 1183) or CGTxxxPPR/K (SEQ ID NO: 1184) presented a higher tropism for astrocytes.

Example 12. Capsid Testing

Capsid variants representative of distinct sequence clusters (highlighted in FIG. 37B) were chosen for individual transduction analysis in C57BL/6 mice. Each capsid was produced as a recombinant AAV packaging a self-complementary EGFP transgene driven by the ubiquitous promoter (FIGS. 49A, B). Mouse groups (N=3) were injected intravenously with 6e10 VG and transduction efficiency was assessed after 1 month by quantifying EGFP mRNA in the brain, spinal cord, and liver tissue. EGFP mRNA expression was normalized using mouse TBP as a housekeeping gene, and DNA biodistribution was normalized to the single-copy mouse TfR gene (FIG. 50A-FIG. 50C). Reverse transcription was performed with the Quantitect kit and included a DNA removal treatment. All capsid variants showed a significant improvement in brain and spinal cord mRNA expression by comparison to the parent AAV9 capsid, and 3 out of 7 variants (9P16, 9P31 and 9P35) showed similar or higher transduction than the PHP.eB benchmark capsid (FIG. 49C, Table 10). The viral DNA biodistribution showed a very strong tropism of 9P31 and 9P35 for the brain and spinal cord, but all the variants showed a 40- to 260-fold increase of biodistribution compared to AAV9 (FIG. 49D, Table 10).

Expected cellular tropism was tested using an NGS screen by labeling the neuronal NeuN marker (FIG. 51). Within the cortex, the top capsids in the GFAP screen showed mostly GFP expression in NeuN-negative cells with glial morphology. Conversely, top capsids in the SYN screen showed a very high transduction of NeuN-positive cells, and the dual-specificity capsids 9P08 and 9P16—ranking high in both assays—showed mixed cell preference with multiple NeuN+ cells and glial cells.

Cellular tropism was also tested using mouse brain microvascular EC (mBMVEC) binding relative to AAV9. Results are shown in Table 9.

TABLE 9 mBMVEC binding results BINDING TO SEQUENCE  mBMVEC (fold PEPTIDE SEQUENCE ID over AAV9) AAV9 AQ   1 PHP.eB DGTLAVPFK  71 153 9P03 AQTTEKPWL  83 170 9P08 DGTLSQPFR  65 349 9P09 DGTMDRPFK 102 222 9P13 AQGENPGRW  96   2.5 9P16 DGTADKPFR  63 176 9P31 AQWPTSYDA  62   2 9P32 DGTAIHLSS  67   16 9P33 DGTGQVTGW  68   5 9P36 DGSSSYYDA  64   0 9P39 DGTGSTTGW 134   2

Fluorescent EGFP expression in tissues of whole brain, cerebellum, cortex, and hippocampus revealed transduction patterns across a spectrum and demonstrate the identification of tissue-specific capsids (FIG. 52-FIG. 56).

The liver transduction, measured by mRNA expression and by whole tissue GFP expression, showed that several variants outperformed AAV9, which was unexpected in light of the NGS results. Some variants, such as 9P08 or 9P23, showed a relative liver detargeting by comparison with AAV9 (FIG. 57A-FIG. 57B).

TABLE 10 Brain and Spinal cord tropism BRAIN EGFP mRNA* EGFP/TBP EGFP/TBP EGFP/TBP group group Mean Fold Fold CAPSID m1 m2 m3 mean SD over AAV9 SDEV AAV9 0.11 0.1 0.15 0.12 0.03 1 0.21 PHPN 2.94 4.44 3.42 3.6 0.77 30 6.38 9P08 2.46 3.47 2.73 2.89 0.53 24 4.38 9P12 3.07 2.27 2.98 2.77 0.44 23 3.65 9P16 4.31 4.75 5.28 4.78 0.49 39 4.06 9P23 3.28 2.37 2.79 2.81 0.46 23 3.79 9P30 1.06 1.7 1.32 1.36 0.32 11 2.66 9P31 4.87 5.53 4.2 4.87 0.66 40 5.54 9P35 3.9 3.24 3.45 3.53 0.33 29 2.78 PHPB*** 2.68 2.68 2.68 2.68 0 22 0 ctrl 0 0 0 0 0 0 0 SPINAL CORD EGFP mRNA* EGFP/TBP EGFP/TBP EGFP/TBP group group Mean Fold Fold CAPSID m1 m2 m3 mean SD over AAV9 SD AAV9 0.84 0.29 0.3 0.48 0.31 1 0.66 PHPN 3.36 5.8 5.4 4.86 1.31 10.22 2.75 9P08 4.3 5.62 4.65 4.86 0.68 10.22 1.43 9P12 6.09 5.94 5.78 5.94 0.16 12.49 0.33 9P16 4.42 5.31 5.37 5.04 0.53 10.6 1.12 9P23 5.41 5.95 5.04 5.47 0.46 11.5 0.96 9P30 1.53 1.83 2.11 1.82 0.29 3.84 0.61 9P31 6.92 7.06 6.94 6.98 0.08 14.68 0.16 9P35 4.68 4.81 4.79 4.76 0.07 10.02 0.15 PHPB 3.84 3.84 3.84 3.84 0 8.09 0 ctrl 0 0 0 0 0 0 0 BRAIN EGFP DNA** (VG/Cell) EGFP/TERT EGFP/TERT EGFP/TERT Group Group Mean Fold Fold CAPSID m1 m2 m3 mean SD over AAV9 SDEV AAV9 0.03 0.04 0.01 0.03 0.01 1 0 PHPN 2.07 2.79 1.94 2.27 0.46 87 18 P08 1.25 1.62 5.47 2.78 2.34 107 90 P12 1.43 0.94 1.41 1.26 0.27 48 10 P16 4.13 1.15 3.56 2.95 1.58 113 60 P23 1.34 2.68 1.87 1.96 0.68 75 26 P30 0.59 1.42 1.21 1.08 0.43 41 17 P31 6.47 5.6 8.81 6.96 1.66 267 64 P35 4.62 5.55 2.52 4.23 1.55 162 59 PHPB 1.5 1.5 1.5 1.5 0 58 0 ctrl 0 0 0 0 0 0 0 SPINAL CORD EGFP DNA** (VG/Cell) EGFP/TERT EGFP/TERT EGFP/TERT Group Group Mean Fold Fold CAPSID m 1 m 2 m 3 AVG SD over AAV9 SDEV AAV9 0.03 0.04 0.04 0.03 0.007 1 0.2 PHPN 1.75 2.96 3.14 2.62 0.752 75 21.7 P08 3.81 3.47 3.66 3.65 0.174 105 5 P12 1.62 3.31 2.87 2.6 0.873 75 25.2 P16 3.3 3.34 2.96 3.2 0.211 92 6.1 P23 2.63 2.47 3.1 2.73 0.322 79 9.3 P30 0.8 1.8 1.43 1.34 0.507 39 14.6 P31 9.88 6.19 5.47 7.18 2.366 207 68.2 P35 2.95 3.92 2.41 3.1 0.765 89 22 PHPB 1.34 1.34 1.34 1.34 0 39 0 ctrl 0 0 0 0 0 0 0 *EGFP mRNA expression was normalized to TBP as a housekeeping marker **GFP DNA was normalized to single-copy TfR DNA ***N = 1

Example 13. Multi-Rodent Testing (Cross Species)

The efficacy of the 333 capsid variants to transduce CNS was tested in other rodent strains or species (FIG. 47). Side-by-side comparison of neuron and astrocyte transduction in C57BL/6 mice, BALB/C mice and rats showed major differences in the enrichment scores of multiple variants between the two mouse strains, and even more pronounced differences between mice and rats (FIG. 48A-FIG. 48C). Strikingly, the most efficient capsid for rat brain transduction was the parental AAV9, which suggests that directed evolution “bottlenecks” capsid variants that are highly performant in one given species, as opposed to the versatility of wild-type AAV capsids.

Correlation analysis showed that some capsids shared high CNS transduction between C57BL/6 and BALB/C mice, whereas others were restricted to only one strain (FIG. 48B).

Interestingly, the PHP.B and PHP.eB capsid showed poor brain transduction in BALB/C mice, in line with a recent publication (Hordeaux et al., 2018). When focusing on the capsids that showed >10-fold increase in brain transduction, 62 variants were improved only in C57BL/6 mice, 28 variants were improved only in BALB/C mice and 30 variants showed improved brain transduction in both strains (Table 11). Consensus sequence analysis showed a “C57BL/6 signature” closely resembling the PHP.eB peptide (DGTxxxPFR (SEQ ID NO: 1185)) whereas the BALB/C signature showed a different consensus (DGTxxxxGW (SEQ ID NO: 1183)), suggesting the use of a different cellular receptor (FIG. 48C).

TABLE 11 TOP 30 candidates from C57BL/6 and BALB/C mouse screen SYNAPSIN PROMOTER C57BL/6 BALB/C REPLICATE 1 (N = 3) REPLICATE 2 (N = 6) REPLICATE 1 (N = 6) Brain Brain Brain Enrichment Enrichment Enrichment 9-mer peptide Factor (fold 9-mer peptide Factor (fold 9-mer peptide Factor (fold insert over AAV9) insert over AAV9) insert over AAV9) DGTSSYYDS 36.40 AQWPTSYDA 39.97 DGTGSTTGW 57.05 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 59) 62) 134) AQPEGSARW 35.95 AQPEGSARW 31.83 DGTGQVTGW 49.87 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 60) 60) 68) DGTASYYDS 32.34 DGTGQVTGW 20.35 DGTGSTHGW 43.08 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 61) 68) 119) AQWPTSYDA 30.81 DGTAIHLSS 19.55 DGTGSTQGW 38.31 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 62) 67) 315) DGTADKPFR 29.30 DGTMDRPFK 19.48 DGTGTTTGW 37.29 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 63) 102) 130) DGSSSYYDA 28.05 DGTGSTTGW 19.20 AQWAAGYNV 34.57 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 64) 134) 245) DGTLSQPFR 26.73 DGSSSYYDA 18.08 DGTGGTKGW 33.59 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 65) 64) 107) DGTAIHLSS 26.23 DGTSSYYDA 17.93 DGTGSTKGW 29.64 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 67) 381) 313) AQFPTNYDS 26.07 DGSQSTTGW 17.59 DGSQSTTGW 25.19 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 66) 136) 136) DGTMDKPFR 25.05 DGTGSTQGW 17.24 AQWEVKGGY 23.44 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 70) 315) 247) DGTLAVPFK 24.62 DGTGTTTGW 17.00 DGTAIHLSS 22.81 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 71) 130) 67) DGTGNVTGW 24.05 DGTLAVPFK 16.84 DGGGTTTGW 22.62 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 69) 71) 270) DGTGQVTGW 23.83 DGTASYYDS 16.68 DGTGGLTGW 22.42 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 68) 61) 294) DGTHIHLSS 22.93 DGTMDKPFR 16.68 DGTNTINGW 20.76 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 162) 70) 124) DGTGNTHGW 22.63 DGTVANPFR 16.32 DGAGGTSGW 19.55 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 72) 394) 151) DGTVIHLSS 22.62 DGTLNNPFR 16.24 DGTNTTHGW 18.99 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 73) 109) 113) DGTLNNPFR 22.33 DGTLAAPFK 15.96 DGTGTVQGW 18.84 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 109) 120) 327) DGTGNTSGW 22.10 DGTLSQPFR 15.43 DGTGQTIGW 18.55 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 137) 65) 305) DGTGTTVGW 21.72 DGTHIHLSS 15.11 AQWELSNGY 18.13 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 74) 162) 246) DGTSSYYDA 20.94 AQTTEKPWL 15.00 DGTGSLNGW 17.93 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 381) 83) 309) DGAGTTSGW 20.42 DGTGNVTGW 14.90 DGTGTTLGW 17.48 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 265) 69) 323) DGGGTTTGW 20.27 DGTGGVTGW 14.89 AQPEGSARW 17.11 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 270) 299) 60) DGTLQQPFR 19.88 DGTSSYYDS 14.80 DGTGSTMGW 16.91 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 89) 59) 314) DGTGQTIGW 19.52 DGTGNTSGW 14.48 DGTGNTHGW 16.47 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 305) 137) 72) DGTVTTTGW 19.49 AQWPTAYDA 14.48 DGSGTTRGW 15.83 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 405) 256) 114) DGTSIHLSS 19.45 AQGENPGRW 14.41 DGTNSTTGW 15.48 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 378) 96) 143) DGTGSTTGW 19.45 DGTADKPFR 14.32 DGRNALTGW 15.13 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 134) 63) 275) DGTGGVTGW 19.44 DGTGQTIGW 14.27 DGAAATTGW 15.02 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 299) 305) 264) DGTVANPFR 19.42 DGTISQPFK 13.84 DGTATTMGW 14.54 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 394) 105) 284) DGTGTTTGW 19.16 DGTKLMLSS 13.71 AQRYTGDSS 14.35 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 130) 157) 138) DGAGGTSGW 18.99 AQTLAVPFK 13.69 DGAGTTSGW 14.29 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 151) 168) 265) GFAP PROMOTER C57BL/6 BALB/C REPLICATE 1 (N = 2) REPLICATE 2 (N = 6) REPLICATE 1 (N = 6) Brain Brain Brain Enrichment Enrichment Enrichment 9-mer peptide Factor (fold 9-mer peptide Factor (fold 9-mer peptide Factor (fold insert over AAV9) insert over AAV9) insert over AAV9) DGTADKPFR 37.60 DGTMDRPFK 24.89 DGTGSTTGW 21.03 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 63) 102) 134) DGTLSQPFR 35.97 DGTAERPFR 24.66 DGTGQVTGW 19.24 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 65) 140) 68) DGTTYVPPR 33.09 DGTADKPFR 23.03 DGTGTTTGW 15.56 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 75) 63) 130) DGTNGLKGW 32.14 DGTLNNPFR 22.91 DGTGSTHGW 14.45 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 76) 109) 119) AQGENPGRW 31.99 DGTLSQPFR 21.60 DGTAIHLSS 11.74 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 96) 65) 67) AQGSWNPPA 30.78 DGTMDKPFR 20.52 DGTGSTQGW 11.40 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 80) 70) 315) AQGTWNPPA 29.19 DGTISQPFK 20.47 DGTGGLTGW 8.87 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 82) 105) 294) DGTISQPFK 29.01 AQGENPGRW 20.09 AQNGNPGRW 8.82 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 105) 96) 84) DGTTFTPPR 28.94 AQTTEKPWL 18.04 DGTGGIKGW 8.62 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 79) 83) 131) DGTRTTTGW 28.59 DGTVANPFR 16.87 DGRNALTGW 8.39 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 106) 394) 275) DGTSYVPPR 26.17 DGTTYVPPR 16.31 DGTGSTKGW 8.38 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 78) 75) 313) DGTIERPFR 25.37 AQTTDRPFL 16.27 AQRYTGDSS 8.13 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 87) 85) 138) DGTMDRPFK 24.85 DGTTTYGAR 15.62 DGTGGTKGW 8.06 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 102) 77) 107) DGTLAAPFK 24.67 DGTADRPFR 15.60 DGTATTTGW 8.04 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 120) 155) 285) DGTLNNPFR 24.62 DGTIERPFR 15.11 DGTKMVLQL 7.87 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 109) 87) 142) DGTSFTPPR 24.14 AQGSWNPPA 15.11 DGTGSLNGW 7.71 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 88) 80) 309) AQTTDRPFL 23.85 AQGTWNPPA 15.03 DGTNTINGW 7.59 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 85) 82) 124) DGTSFTPPK 23.75 DGSTERPFR 15.01 AQWELSNGY 7.57 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 81) 99) 246) DGTHTRTGW 23.54 AQSVAKPFL 14.90 DGTNGLKGW 7.50 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 90) 231) 76) DGTLQQPFR 22.94 DGTVDRPFK 14.74 DGTNTTHGW 7.25 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 89) 395) 113) AQNGNPGRW 22.80 DGTTFTPPR 14.56 DGTRMVVQL 7.25 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 84) 79) 370) DGTAERPFR 21.65 AQTLARPFV 14.51 DGTNSTTGW 6.41 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 140) 98) 143) DGTGNTRGW 21.12 DGTGGTKGW 14.13 DGSQSTTGW 6.29 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 94) 107) 136) AQTTEKPWL 20.58 AQGPTRPFL 13.47 AQPEGSARW 6.23 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 83) 125) 60) DGTADRPFR 20.49 DGTRTTTGW 13.39 DGTGQTIGW 6.16 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 155) 106) 305) DGTTWTPPR 20.44 AQNGNPGRW 13.09 DGTGGVTGW 6.07 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 139) 84) 299) DGTTTYGAR 20.43 DGTVSNPFR 12.77 DGTVTTTGW 6.04 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 77) 403) 405) DGTGGIKGW 20.20 AQGGNPGRW 12.21 DGKGSTQGW 5.97 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 131) 91) 272) DGTLAVPFK 19.43 AQWPTSYDA 11.93 AQGENPGRW 5.88 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 71) 62) 96) DGKQYQLSS 18.74 DGTLQQPFR 11.92 DGNGGLKGW 5.82 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 92) 89) 167) DGSPEKPFR 18.73 DGTNGLKGW 11.53 DGTGTVHGW 5.82 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 160) 76) 326)

The efficacy of the 333 capsid variants to transduce CNS was also compared for C57BL/6 mice BMVEC and Human BMVEC (FIG. 58A and FIG. 58B).

Example 14. Engineering of a NGS-Driven Selection System for Full-Length Capsid Variants

A barcode system was engineered to allow enrichment studies with full capsid length modifications. While the TRACER platform described here was initially developed for the use of peptide display libraries, where the randomized peptide sequence itself can be used for Illumina NGS analysis due to its short size, the Illumina sequencing technology does not typically allow sequencing of more than 300 contiguous bases, and therefore our platform cannot be used for NGS analysis of full-length capsid variants, such as those generated by DNA shuffling technology or error-prone PCR.

An alternative RNA-driven platform for full-length capsid libraries in which a unique molecular identified (UMI) is placed outside the capsid gene and can be used for NGS enrichment analysis was designed (FIG. 59A-FIG. 59C). Once the variants with desired properties are identified by UMI enrichment analysis from animal tissue, the UMI sequence must allow highly specific recovery of the full-length capsid from the starting material with a minimal error rate. The system should have one or more of the following properties to be effective: 1) the UMI should be transcribed under control of a cell type-specific promoter, 2) the UMI should not interfere with capsid expression or splicing during virus production, 3) the UMI should be short enough for Illumina NGS sequencing (typically less than 60nt for standard single-end 75 nt sequencing), and 4) the UMI should allow sequence-specific recovery of full-length capsids of interest from the starting DNA/virus library with a minimal error rate.

To address these properties: 1) the UMI was placed in the transcribed region of capsid library (i.e., anywhere between the transcription start site and the polyadenylation signal), 2) the UMI was placed either in various locations of the AAV intron (which mostly unspliced in the absence of helper functions) or between the capsid stop codon and the polyadenylation signal, 3) the UMI cassette was composed of two randomized 21-nt sequences separated by a 15-nt spacer, for a total length of 57 nt, which allows 18 extra nucleotides for primer annealing, and 4) the UMI randomized sequences were formed of NSW triplets (N=A, T, G, C; S=G, C; W=A, T) to prevent large variations in annealing temperature with amplification primers, avoid homopolymeric stretches and prevent the formation of a premature polyA signal (AATAAA).

Importantly, the UMI cassette contained two random sequences in tandem. The first sequence (outermost) is used to design a matching capsid recovery primer, and the second sequence (innermost) to confirm the identity of the capsid amplicon after cloning. This method should allow to eliminate all clones containing non-specific amplification products. In an alternative embodiment, the innermost sequence can also be used to design a nested PCR primer in order to increase the specificity of amplification (FIG. 59A-FIG. 59C).

Several insertion sites of the tandem barcode to test the impact on virus viability and titers were explored. A series of constructs were engineered with the barcode inserted in the AAV intron of the CAG9 plasmid (FIG. 60A). Since AAV intron is spliced during virus production, the presence of the barcode should have only a minimal impact on the yields. Conversely, the AAV splicing is very ineffective in the absence of helper functions (Mouw et al., 2000), therefore the barcode sequence will be preserved in the RNA recovered from animal tissue. All intronic barcode constructs were tested for their ability to produce high titer AAV progeny by cotransfecting them with pHelper and pREP3 stop plasmids. All constructs allowed high titer AAV production going from 50% to 80% of non-barcoded CAG9 virus (FIG. 60B).

RNA splicing analysis from transfected cells showed that the rate of AAV intron splicing was slightly different between constructs and was more efficient when the intronic barcode was inserted after a conserved intervening sequence downstream of the splice donor (FIG. 58C, upper panel).

Globin intron splicing was 100% effective in all tested conditions (FIG. 60C, lower panel). As expected, AAV intron splicing was almost undetectable in the absence of helper functions.

An alternative platform was tested where the tandem barcode was placed between the capsid stop codon and the polyadenylation signal (FIG. 59B). Titers produced by the 3′-barcoded constructs were identical to the non-barcoded CAG9 construct.

Overall, external barcoding of full-length capsid allows highly efficient AAV production, and the novel tandem barcode platform allows NGS-driven sequence-specific recovery from library preparations with high confidence.

TABLE 12 Sequences DESCRIPTION SEQ ID NO: NUCLEIC ACID SEQUENCE PREP2 SEQ ID CGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTA NO: 4 CGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAG CTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCT GAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGAC GGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGAAGGG AGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTT GGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGA GCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGA ACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGC TCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGC GTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAA GAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTAC ATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCA GGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAA GGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCT GGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACT AAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTT CGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCG CGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACT TTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACC GCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCA GAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAA CATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGA CCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAA GCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATG AATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATA AGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGC TTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCT GATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCAC TCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTC GTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGAC GCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA ATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTC TCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGC AGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGAC CCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAG CACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAA CCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCT CGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTCCACCA TACCTTCGATTATCCGATTTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGCGGCCGCCACCGCGGT GGAGCTCCAGCTTTTGT CMV9-BSTEII TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC SEQ ID NO: 5 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCGTTTAAACC GCGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT TACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACG GGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCG TGTACGGTGGGAGGTCTATATAAGCAGAGCTCGGGAGCGGTCACCAAGCAGGAAGTCAA AGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAA AAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAAC GGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTAC GCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAA GACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGT ATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTT GCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCA GGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATT CGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCA AGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACT CGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCT ACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCC GAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGT CTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGAC GGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGG GTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCG ACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTG TGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTG CCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACA GAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACA AGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACA GCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTG GCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTT CAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACC TTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGT CGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACG GGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGG AATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTG AGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATC CACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATC AACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAAC TACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAAC AACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGA TGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGT CTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAA GTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTA TGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTC AAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGA CCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGA GGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCG GATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACT GGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGA ACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAA TACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCT GTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGT ATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTA ACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCA CTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA PREP-AAP GTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC SEQ ID NO: 6 CGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCC GGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCT GACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGAC TTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGA AGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGC CGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAG GTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGG CGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGT GGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCA ATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGC TCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCT CCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGA TTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGT CTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGC AACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGT AAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGA GGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGG TGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCT CCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGT TGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCAC CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAA CTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGAC TGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGA AACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGT CAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA TGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTG AAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTG CTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCA GCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAA CCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGT CTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAG GAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCA GCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAAT AACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTG GGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTC TACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTAC AGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGC AGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACAT TCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCAC GGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGC TGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATG ATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCT AAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTAC GCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCT CAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCT CAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCT CAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAAGTCAGCGTGGAGATCGAG TGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTAT TACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCA TTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTT TCAGTTGAACTTTGGTCTC PREP3 STOP GTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC SEQ ID NO: 7 CGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCC GGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCT GACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGAC TTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGTGCAATTTGAGA AGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGC CGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAG GTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGG CGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGT GGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCA ATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGC TCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCT CCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGA TTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTT CCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGT CTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGC AACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGT AAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGA GGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGG TGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCT CCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGT TGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCAC CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGA ATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAA CTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCC TGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGAC TGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGA AACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGT CAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA TGGTTAGCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTG AAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTG CTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCA GCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAA CCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTC TTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGT CTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGTAGAGGCCTGTAGAGCAGTCTCCTCAG GAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAAT TTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCA GCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAAT AACTAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTG GGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTC TACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTAC AGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGC AGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACAT TCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCAC GGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGC TGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATG ATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCT AAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTAC GCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCT CAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCT CAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCT CAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAAGTCAGCGTGGAGATCGAG TGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTAT TACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCA TTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATTCGTT TCAGTTGAACTTTGGTCTC SYN-CAP9 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC SEQ ID NO: 8 GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT CCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCTAGTATCTGCAGAGGGCCCT GCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGA CCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGA GAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGA CAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGA CGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGC CGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGC GCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGT GCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGAC CACCCCTAGGACCCCCTGCCCCAAGTCGCAGCCGGTCACCAAGCAGGAAGTCAAAGACTTTTT CCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGC CAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAG TTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACA AATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAA TCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCA GAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCA TGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGT TTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA GGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAA GGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGG ACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGC ACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACG CCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAG CAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGA CGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTA TTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAG AGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCT TACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGG TAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAG CACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCAC ATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGAC TTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGG GATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACA ACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAG ACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGA CGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGT TCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGT TCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCG ACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGA CAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGA AACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAAC AACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGA ATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATC TTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAAC CAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCAC AAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCC GGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCA CACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCT CAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAG CTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGC AGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTA ATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAG ATACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAAC TTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGG TTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCT CGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA GFAP-CAP9 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGAC SEQ ID NO: 9 GCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT CCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCGATCTAACATATCCTGGTGTG GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGG AGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGG GCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACA GTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGG GGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTA GGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCC AGGAAAGGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGG CTGTCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGG GCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCAT CGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAG GTTGGAGAGGAGACGCATCACCTCCGCTGCTCGCGGGGATCCTCTAGGGTCACCAAGCAGGAA GTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTC AAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACG GGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGA CAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAA TGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGT GCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTA CATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGAC TTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTT CCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGA GCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGT TACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGC GGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCT CAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGG CAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAG GAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGA CTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCA GACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTC AGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGG TGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAG AGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCA AATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCC TGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCA TCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAA AGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGT CTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCG CCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCC AGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGG TAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGC CAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTA TTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGG CTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTG TGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACG TAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTT CCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGAC AAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTAT GGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAA CCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTG GGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATG AAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCT TCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGAT CGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCA ACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCG CCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAA TTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGAT AAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTG CCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA CAG-CAP9 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC SEQ ID NO: 10 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG ACATAACGCGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATT AGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGG CTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTT GGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCACTCTCCCCAT CTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGA TGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGC GGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG GAGCAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGA CCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAA CGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTC TATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAAT ACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCAC CATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATAT AAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTA CAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTC CAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGG CAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGT CACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGG AGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCA GATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGC GGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCAT GAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTG CTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTT TCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTG CCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAAC AATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGAC AACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAG GCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTT GGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCT CGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGT ACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGC AACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTT GAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGA ACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCA ATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTC CCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGG CAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATT CCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCT ACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACA ACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCA CTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCG ACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAA GACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCT CCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTT CATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTC GTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCA GTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCT GGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAA CGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGG CTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCA CTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCA ATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAG GACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACA ACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCG GTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGC GCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAG ATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACC CTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAA ACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCA TCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAA AACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAAT GTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGA TACCTGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTG AACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCAT GGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTG CGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCC CGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA SYNG-CAP9 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC SEQ ID NO: 11 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG ACATAACGCGTGATCTAACATATCCTGGTGTGGAGTAGCGGACGCTGCTATGACAGAGGC TCGGGGGCCTGAGCTGGCTCTGTGAGCTGGGGAGGAGGCAGACAGCCAGGCCTTGTCTG CAAGCAGACCTGGCAGCATTGGGCTGGCCGCCCCCCAGGGCCTCCTCTTCATGCCCAGTG AATGACTCACCTTGGCACAGACACAATGTTCGGGGTGGGCACAGTGCCTGCTTCCCGCCG CACCCCAGCCCCCCTCAAATGCCTTCCGAGAAGCCCATTGAGCAGGGGGCTTGCATTGCA CCCCAGCCTGACAGCCTGGCATCTTGGGATAAAAGCAGCACAGCCCCCTAGGGGCTGCCC TTGCTGTGTGGCGCCACCGGCGGTGGAGAACAAGGCTCTATTCAGCCTGTGCCCAGGAAA GGGGATCAGGGGATGCCCAGGCATGGACAGTGGGTGGCAGGGGGGGAGAGGAGGGCTG TCTGCTTCCCAGAAGTCCAAGGACACAAATGGGTGAGGGGAGAGCTCTCCCCATAGCTGG GCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGG CATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCA GAGCAGGTTGGAGAGGAGACGCATCACCTCCGCTGCTCGCGGGGATCCTCTAGAAGCTTC GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAA GACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGG AACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCAC AAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAAT CTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAA TAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCA TATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTAC CATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCC TTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTC TGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCACCGGTCAC CAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGC ATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGAT ATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGA AGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAA TCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTT CACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCT GTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCA GACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAAT AAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAAC CTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCA AATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGA CCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGA GCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACA ACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAAC CTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAG GAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACC GGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTT CGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCG CAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAG ACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCC AATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTAC AACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAAC GCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACT TCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGAC TCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGA CCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCC CGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCA TGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGT CCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTT CAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGA CCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGT TCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTC CAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGT GACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGG ACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACC GTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGT GGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAG CAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAG ACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGT GTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTC TCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACAC ACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCAC CCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACA GCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTG AATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACC TGACTCGTAATCTGTAATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA GFAPG-CAP9 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC SEQ ID NO: 12 CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGT GGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGTCGTGACGATATCCATGCGTCG ACATAACGCGTTAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACC AGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCA CCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGA TGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGC CTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTC CCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCG GACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGC GCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTG AGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGACCA CCCCTAGGACCCCCTGCCCCAAGTCGCAGCCAAGCTTCGTTTAGTGAACCGTCAGATCGC CTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCT CCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAG AGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATG ATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTA AGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAG AGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTT GGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCT CTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTG GCAAAGAATTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTT TTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGG GTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTG CGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGA CAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAG ACAATGCGAGAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTG TTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAG AAACTGTGCTACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGAC CTGGTCAATGTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTAT GGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGA GTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACA ACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACA AGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGAC CAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTT CCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCA GGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCC TGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTG GCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACA GAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGA TCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGAT GGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTC ATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAA ATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACC CCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGC GACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACA TTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACC AGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCT CACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATC TGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTT CCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGT ACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCAT CGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAAC GCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATAC CTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGC GAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATC CTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGAT CTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATG ATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACA AGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACC AAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTT GGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTG GAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTC CAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAG TCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGA GATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAA GGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATCG ATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTT CTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAA GGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGC CGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGAGAGGGAGTGGCCAA GLOSPLICEF6 GTGCCAAGAGTGACCTCCTG SEQ ID NO: 13 CAP5L8 ACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATG GBLOCKSEQ GAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCA ID NO: 14 CTTTCACCCCTCTCCGGCTATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTC ATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGC TTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAA GGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCC AGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGA ACCCGATACCTTACCCGACCCCTTTAA CAP6L8 ACCGGAGATGTGCATGTTATGGGAGCCTTACCTGGAATGGTGTGGCAAGACAGGGACGT GBLOCKSEQ CTACCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCATCT ID NO: 15 CCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACG CCTGTTCCTGCGAATCCTCCGGCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCC AGTATTCCACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGC AAACGCTGGAATCCCGAAGTGCAATATACATCTAACTATGCAAAATCTGCCAACGTTGAT TTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTCA CCCGTCCCCTGTAATCGAT CAPDJ8L8 ACACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAG GBLOCKSEQ GACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACA ID NO: 16 TTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCGCCTCAGATCCTG ATCAAGAACACGCCTGTACCTGCGGACCCTCCGACCACCTTCAACCAGTCAAAGCTGAAC TCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAG AAGGAAAACAGCAAGCGCTGGAACCCCGAGATCCAGTACACCTCCAACTACTACAAATC TACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAACCCCGCCCCATTGG CACCCGTTACCTCACCCGTAATCTGTAA CAP9L8M GCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCA GBLOCKSEQ GGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCA ID NO: 17 ACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCT CATCAAAAACACACCTGTACCTGCCGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAA CTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCA GAAGGAAAACAGCAAGCGGTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGT CTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGG CACCAGATACCTGACTCGTAATCTGTAA TELN-SYNG9- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC BSRGI SEQ ID TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG NO: 18 TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC CGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAG GTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGA CGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAG ACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGG GCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGACTGAATCAGAATTCAAATA TCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCACATCATGGGAAA GGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAATGTGGACTTGGATGACTGTGTTTCT GAACAATAAATGACTTAAACCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA GGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACC CAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATA CCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGG CCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTC AAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGG GGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCT GGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTC AGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGA CTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAA CCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCA GTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTG CGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCC CACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAA TGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCA CTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCC TAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGG AGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTA TCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGA CGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGG TCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAAC TTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAA AGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACT ATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAA CATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTC AACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGC TCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGG AGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGA GACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAA CCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGTACATCGAT TGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCT TATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGG AACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA GCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTG ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCA GCTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA TELN- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC GFAPG9- TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG BSRGI SEQ ID TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT NO: 19 GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA GAGACTGAATCAGAATTCAAATATCTGCTTCACTCACGGTGTCAAAGACTGTTTAGAGTG CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCACATCATGGGAAAGGTGCCAGACGCTTGCACTGCTTGCGACCTGGTCAAT GTGGACTTGGATGACTGTGTTTCTGAACAATAAATGACTTAAACCAGGTATGGCTGCCGA TGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGC TTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAG GTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGC CGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTC AAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCG GCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGA AGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGG GTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTC CCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACA ATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGG TAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCAC CAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAA CAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGG GTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATC AACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTC AAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGT CCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGG CTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTT AATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGC AAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCC ATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAAT ACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAAT TCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCC AGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCT TGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTG CTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTT TGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACG AAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACA AACCACCAGAGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACT TTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTG GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT GCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTAT AATGGACTATTGTGTGCTGATA TELN-SYNG5- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC BSRGI SEQ ID TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG NO: 20 TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT GAACAATAAATGATTTAAATCAGGTATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAA GAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAA CCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTC GGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCG AGAGCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGT ACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGA AACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTT GAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAG AAAGAAGGCCCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTG GACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTG ATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGA GTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTC ACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGAT CAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTG GGGGTACTTTGACTTTAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACT CATCAACAACTACTGGGGCTTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCA AGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCAC CGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGA GGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGAC GCTGAACCGCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGT ACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGG AGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTCTTCAAGCTGGCCAACCCGCT GGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAA CAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGG GCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCG CCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGAAC GGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTC AACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCAT CACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGA TGGCCACCAACAACCAGAGCTCTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTT TCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAA GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCC CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATG CAATTAACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATA CGCGCGTATAATGGACTATTGTGTGCTGATA TELN- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC GFAPG5- TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG BSRGI SEQ ID TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT NO: 21 GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGTCTTTTGTT GATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTT GAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCT TGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGT CAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCTTGAGG CGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTC GCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAG GGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCG GATAGACGACCACTTTCCAAAAAGAAAGAAGGCCCGGACCGAAGAGGACTCCAAGCCTT CCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCC AACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCG ACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCC ACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTA CAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACG CCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACAGCCACTG GAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCCCT CAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCAC CATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCC CTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTAC GCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAGA GGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACT TTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAA CCTCTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAA TAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACA AAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTC AACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAG TTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCT ATGCCCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCC ACGTACCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCG CGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCTGTACATCGATT GTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTT ATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGA ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG CGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTGA CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA TELN-SYNG6- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC BSRGI SEQ ID TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG NO: 22 TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT GAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA GGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAAC CCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAG TACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGATGCAGC GGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACC TGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTG GGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTC TGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCA CAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAG ACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGA ACCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCGCACC AATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATT GCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGC CCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACG ACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTG CCATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAA GAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGT CACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCA GTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTG TTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGG TCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTA CCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCC TGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGAACAGAACTCAGA ATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCA TGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTA AAACAAAAACAGACAACAACAACAGCAACTTTACCTGGACTGGTGCTTCAAAATATAAC CTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCCTCACACAAAGACGAC AAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCT TCAAACACTGCATTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAGCCACTAA CCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGTCAATCTCCAGAGTGTACATCGATT GTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTT ATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGA ACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGG GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG CGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACAACGTCGTGA CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGTGCTGATA TELN- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC GFAPG6- TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG BSRGI SEQ ID TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT NO: 23 GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGA TGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGA CTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGG GTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGC CCGTCAACGCGGCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTC AAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCG TCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAA GAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAA ACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAG GCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCC CCGACCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAA TGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGT AATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACC AGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGT GCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTAT TTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTCATCAACA ACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGG AGGTCACGACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAG TCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCT CCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAA TGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGAT GCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAG CAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCT GTATTACCTGAACAGAACTCAGAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTT TAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTG TTACCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAACTTTACCT GGACTGGTGCTTCAAAATATAACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTG CTATGGCCTCACACAAAGACGACAAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTT TTGGAAAGGAGAGCGCCGGAGCTTCAAACACTGCATTGGACAATGTCATGATCACAGAC GAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGT CAATCTCCAGAGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAAC TTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGC GGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG GCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTG GCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTT GCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTAT AATGGACTATTGTGTGCTGATA TELN- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC SYNGDJ8- TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG BSRGI SEQ ID TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT NO: 24 GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTTAGTATCTGCAGAGGGCCCTG CGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGA CGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCC TATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCA GCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACT GAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGT CGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGG GCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGT GGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCA GTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCC AAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTG CATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAG GCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTC CCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCT AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATAT TTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCC AGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCT AGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACCGGTCGCCAC CGGTCACAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGG TGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGA CGCGGAAGCTTCGATCAACTACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATAT CTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAA GGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTT GAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA GGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCAC CAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAG TACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGC GGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACC TCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTG GGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTC TGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCACTCTCCT GTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAG ATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTCCCAGACCCTCAACCAATCGGAGA ACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTGCAGGCGGTGGCGCACC AATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATT GCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGC CCACCTACAACAACCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAA ATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCC ACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGC CCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAA GGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAG TACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGG ACGTGTTCATGATTCCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGG GACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCGCAGATGCTGAGAACCGGCAACA ACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCCCACAGCCA GAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGGACT CAAACAACAGGAGGCACGACAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAA TACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAGCAGCGAG TATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAG TACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAG GACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCA GAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGAC AACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGCAAGGTGT ACATCGATTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTA TTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAA CTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATGCAATTAACTGGCCGTCGTTTTACA ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC TTTCGCCAGCTGTATCAGCACACAATTGCCCATTATACGCGCGTATAATGGACTATTGTGT GCTGATA TELN-GFAPG- TATCAGCACACAATAGTCCATTATACGCGCGTATAATGGGCAATTGTGTGCTGATACAGC DJ8-BSRGI TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG SEQ ID NO: 25 TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAG ATTTAATTAAGGCCTTAATTAGGCTAGCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTC ACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGT GAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGG GTGGAGTCGTGACGATATCCATGCGTCGACATAACGCGTGATCTAACATATCCTGGTGTG GAGTAGCGGACGCTGCTATGACAGAGGCTCGGGGGCCTGAGCTGGCTCTGTGAGCTGGG GAGGAGGCAGACAGCCAGGCCTTGTCTGCAAGCAGACCTGGCAGCATTGGGCTGGCCGC CCCCCAGGGCCTCCTCTTCATGCCCAGTGAATGACTCACCTTGGCACAGACACAATGTTC GGGGTGGGCACAGTGCCTGCTTCCCGCCGCACCCCAGCCCCCCTCAAATGCCTTCCGAGA AGCCCATTGAGCAGGGGGCTTGCATTGCACCCCAGCCTGACAGCCTGGCATCTTGGGATA AAAGCAGCACAGCCCCCTAGGGGCTGCCCTTGCTGTGTGGCGCCACCGGCGGTGGAGAA CAAGGCTCTATTCAGCCTGTGCCCAGGAAAGGGGATCAGGGGATGCCCAGGCATGGACA GTGGGTGGCAGGGGGGGAGAGGAGGGCTGTCTGCTTCCCAGAAGTCCAAGGACACAAAT GGGTGAGGGGAGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTA TGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAA GCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGTTGGAGAGGAGACGCATCACCT CCGCTGCTCGCGGGGATCCTCTAGAAGCTTCGTTTAGTGAACCGTCAGATCGCCTGGAGA CGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGA TTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGT AAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAATATACTTTT TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAAT GTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAAT AGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCA TATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAG GCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTC CTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAA TTGGGATTCGAACCGGTCGCCACCGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTG GGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCA GTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCGGACAGGTACCA AAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGA GAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTG CTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGA TGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAA GCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGG GTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGC CGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTC GACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCG GCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAA AGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGA AGAGGCCTGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCG GGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGT CCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTAC AATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGG GTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCA CCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCA ACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGG GGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCAT CAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGT CAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCA TCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGG GCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAACACT CAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTTCG CAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTC CACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAG TACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGACAAATACGCAGACTCTG GGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGG ACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAAT ACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGG GCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTT CTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGAT TACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTG TATCTACCAACCTCCAGCAAGGTGTACATCGATTGTTAATCAATAAACCGTTTAATTCGTT TCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAA GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCC CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGCATG CAATTAACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGTATCAGCACACAATTGCCCATTATA CGCGCGTATAATGGACTATTGTGTGCTGATA

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1. A method for generating a variant AAV capsid polypeptides, wherein relative to a parental AAV capsid polypeptide said variant AAV capsid polypeptides exhibit at least one of improved transduction or increased cell or tissue specificity, said method comprising: a) generating a library of variant AAV capsid polypeptides, wherein said library comprises i) a plurality of capsid polypeptides having a region of randomized sequence of 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids, or ii) a plurality of capsid polypeptides from more than one parental AAV capsid polypeptide; b) generating an AAV vector library by cloning the capsid polypeptides of libraries (i) or (ii) into AAV vectors, wherein said AAV vectors comprise a first promoter and a second promoter, wherein said second promoter drives capsid mRNA expression in the absence of helper virus co-infection.
 2. The method of claim 1, wherein the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.
 3. The method of claim 1, wherein the first promoter is AAV2 P40 and the second promoter is a cell-type-specific promoter.
 4. The method of claim 2 or claim 3, wherein the promoter is selected from any of those listed in Table
 3. 5. The method of claim 4, wherein the ubiquitous or cell-specific promoter allows the expression of RNA encoding the capsid polypeptides.
 6. The method of claim 5, further comprising the recovery of said RNA encoding the capsid polypeptides and determining the sequence of said capsid polypeptides.
 7. The method of claim 6, wherein the capsid polypeptides recovered exhibit increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.
 8. The method of claim 7, wherein the target cell is a neuronal cell, a neural stem cell, an astrocyte, an oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell.
 9. The method of claim 1, wherein said AAV vectors comprise a first promoter and a second promoter, wherein said second promoter is located at the downstream of the capsid gene and drives its anti-sense RNA expression in the absence of helper virus co-infection.
 10. The method of claim 9, wherein the first promoter is AAV2 P40 and the second promoter is a ubiquitous promoter.
 11. The method of claim 9, wherein the first promoter is AAV2 P40 and the second promoter is a cell-specific promoter.
 12. The method of claim 10 or claim 11, wherein the ubiquitous or cell-specific promoter allows the expression of gene encoding the capsid polypeptide of variant AAV in an anti-sense direction, resulting in the anti-sense RNA.
 13. The method of claim 12, wherein said method further comprises the recovery of said anti-sense RNA that can be converted to RNA encoding said variant AAV capsid polypeptide that is used to determining the sequence of said variant AAV capsid polypeptides.
 14. The method of claim 13, wherein said variant AAV capsid polypeptide exhibits increased target cell transduction or target cell specificity (tropism) as compared to a parental capsid polypeptide.
 15. The method of claim 14, wherein the target cell is a neuronal cell, a neural stem cell, an astrocyte, a oligodendrocyte, a microglia cell, a retinal cell, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium cell, an endothelial cell, a liver cell, a skeletal muscle cell, a muscle stem cell, a muscle satellite cell, or a cardiac muscle cell. 